Protein modulators

ABSTRACT

The present invention relates to modulators of the Neuregulin (NRG) family, particularly NRG1 and more particularly NRG1β, and most particularly NRG1β1. The present invention also relates to the use of such modulators to inhibit goblet cell hyperplasia and therefore also relates to the use of such modulators in the treatment or prevention of human diseases and disorders featuring pathological mucus production such as COPD, CF, chronic bronchitis and asthma.

1. FIELD OF THE INVENTION

The present invention concerns compounds, compositions and methods forneutralising the biological activity of members of the Neuregulin (NRG)family in the treatment of diseases and disorders. In particular theinvention concerns antagonists which bind with and neutralise thebiological activity of Neuregulin-1 (NRG1) and isoforms thereof in thetreatment of human diseases and disorders. Other aspects, objects andadvantages of the present invention will be apparent from thedescription below.

2. BACKGROUND OF THE INVENTION

Airway mucus hypersecretion has been linked to several of thepathological features of respiratory diseases such as asthma [Aikawa etal, 1992], chronic obstructive pulmonary disease (COPD) [Vestbo, 2002]and cystic fibrosis (CF) [Boucher, 2002]. Indeed, mucus hypersecretionhas been linked to an increase in frequency and duration of infection,decline in lung function and increase in morbidity and mortality inrespiratory diseases [Vestbo, 2002; Prescott et al, 1995; Vestbo et al,1996]. Whilst in the large airways mucus is produced by goblet cells andsubmucosal glands, in the small airways the only source of mucus is thegoblet cell [Rogers, 2003]. The mucins MUC5AC and MUC5B are majorcomponents of airway mucus secretions in respiratory diseases such asasthma, COPD and CF [Williams et al, 2006; Rose and Voynow, 2006;Rogers, 2003]. Mucus hypersecretion is a feature of asthma wheremorphometric analysis of lungs from patients who died from a severeacute asthma attack showed increases in goblet cell numbers and mucus inthe airway lumen [Aikawa et al, 1992]. Mucus plugging of the airwaylumen has been reported as a major contributing cause to fatal asthma inmost patients [Kuyper et al, 2003; Hays and Fahay, 2003]. MUC5ACexpression is increased in status asthmaticus compared to normalindividuals and is localized to the surface epithelium, lumen and gobletcells [Gronenberg et al, 2002a]. Increased numbers of goblet cells havealso been reported in subjects with mild to moderate asthma compared tohealthy individuals and levels of secreted mucin are reported to behigher in the airways of patients with moderate asthma [Ordonez et al,2001]. Additionally, increased MUC5AC mucin staining of goblet cells ofsubjects with asthma compared to healthy individuals has bee reported[Ordonez et al, 2001]. The mucin MUC5B is also produced by some airwaysurface goblet cells in asthmatics [Gronenberg et al, 2002a]. Theprogression of COPD has been reported to be strongly associated withaccumulation of mucus in the lumen of the small airways [Hogg et al,2004]. In individuals with COPD increased expression of MUC5AC has beendescribed within the bronchiolar epithelium in addition to increasedlevels of MUC5B within the bronchiolar lumen [Caramori et al, 2004].MUC5B has also been reported as a major mucin in sputum of patients withCOPD in a separate study [Kirkham et al, 2002]. The increased mucusobserved in the lumen of bronchioles in COPD patients has been suggestedto contribute to obstruction of the peripheral airways in COPD [Caramoriet al, 2004]. Increased numbers of goblet cells in the bronchiolarepithelium of patients with COPD and chronic bronchitis have also beendescribed [Saetta et al, 2000]. In CF mucus hypersecretion is associatedwith airflow obstruction and, in fatal cases, occlusion of the smallairways [Williams et al, 2006]. Excessive mucus also appears tocontribute to CF morbidity by increasing the frequency and severity ofpulmonary infections [Williams et al, 2006]. Although concentrations ofsecreted mucins MUC5AC and MUC5B have been reported to be decreased inCF subjects compared with normals [Henke et al, 2004], MUC5AC and MUC5Bare increased in sputum of CF patients during exacerbations [Henke etal, 2007]. Goblet cell hyperplasia resulting from increased numbers ofMUC5AC-positive cells, have been reported to be increased in cysticfibrosis lung [Gronenberg et al, 2002b].

Whilst I L-13 has been shown to influence MUC5AC gene and proteinexpression in vitro and in vivo [Wills-Karp et al, 1998; Zhu et al,1999; Kuperman et al, 2002; Atherton, Jones and Danahay, 2003], it hasno effect on the mucin MUC5B. The mediators of MUC5B production are notwell characterized.

Neuregulins are signalling proteins that mediate multiple cell-cellinteractions via the receptor tyrosine kinases of the ERB family. Atleast 15 different isoforms of Neuregulin-1 (NRG1) exist as a resultalternative splicing [Falls, 2003]. Two of these isoforms, NRG1a andNRG1β1, differ in the C-terminal portion of the EGF-like domain [Holmeset al, 1992]. NRG1 is thought to bind to ErbB3 or ERRB4 which formheterodimers with ErbB2 [Falls, 2003]. NRG1β1 binds to ErbB3 with100-fold higher affinity than NRG1a. NRG1β1 also has a 100-fold greateraffinity for the ErbB2/ErbB3 heterodimer than ErbB3 homodimers [Jones etal, 1999]. ErbB3 lacks tyrosine kinase activity, but dimerisation withErbB2 results in the formation of an active heterodimer which canmediate downstream signals [Citri, Skaria and Yarden, 2003]. A role forNRG1 and the ErbB2 and 3 receptors in human lung development havepreviously been suggested through immunohistochemical and functionalstudies on fetal lung tissue [Patel et al, 2000]. NRG1β1 is secretedfrom fetal lung fibroblasts and stimulates type II cell surfactantsynthesis and is therefore proposed to control fetal lung maturationthrough mesenchymal-epithelial interactions [Dammam et al, 2003]. Morerecently the NRG1a isoform has been suggested to play a role inepithelial wound repair and remodeling in the airways [Vermeer et al,2003]. In this study the ErbB2 receptor was shown to been expressed onthe basolateral surface of differentiated epithelial cells and NRG1aligand expressed at the apical surface. Consequently ligand receptorinteractions are not thought to take place until an epithelial injuryhas occurred. Expression of NRG (sometimes referred to as Heuregulin,“HRG”) has been examined in bronchial tissue from COPD patients andhigher expression observed in intact epithelium of subjects with COPDcompared to those without COPD [de Boer et al, 2006]. However, in thestudy of de Boer et al. the specific isoform of NRG1 investigated wasnot stated.

All publications, including patent applications, cited in the presentspecification and any specification from which the present applicationclaims priority are expressly incorporated herein by reference in theirentirety.

3. SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the observationthat NRG-1, and in particular the isoform NRG1β1 promotes MUC5AC andMUC5B protein expression and therefore suggests a role in goblet cellformation.

Accordingly, in a first aspect of the present invention there isprovided a method of inhibiting goblet cell formation which methodcomprises providing a binding compound which neutralizes the biologicalactivity of NRG-1, e.g. NRG113 isoform, particularly NRG1β1 (and inparticular human NRG1β1).

In a second aspect of the present invention there is provided a methodof screening for a binding compound capable of neutralizing NRG-1 e.g.NRG1β1 biological activity.

In a third aspect of the present invention there is provided apharmaceutical composition comprising (or consisting essentially of) abinding compound which neutralizes NRG-1, e.g. NRG1β1 (particularlyhuman NRG1β1) biological activity.

In a fourth aspect of the present invention there is provided a methodof inhibiting goblet cell formation in a mammalian patient which methodcomprises administering to said patient a therapeutically effectiveamount of a binding compound which neutralizes the biological activityof NRG-1, e.g. NRG1β1.

In a fifth aspect of the invention there is provided a method ofinhibiting deleterious mucus production in a mammalian patient inclinical need thereof which method comprises administering to saidpatient a therapeutically effective amount of a binding compound whichneutralizes the biological activity of NRG-1, e.g. NRG1β1.

In a sixth aspect there is provided a method of treating a disease ordisorder selected from the group consisting of; COPD, CF, chronicbronchitis and asthma (particularly moderate to severe asthma) in ahuman patient which method comprises administering to said patient atherapeutically effective amount of a human or humanized antibody (ofe.g. a IgG isotype such as IgG1 or IgG4) which neutralizes thebiological activity of NRG1β1 (by for example binding with NRG1β1 andinhibiting the interaction between NRG1β1 and ErbB2/Erb B3 heterodimer).

These and other aspects of the present invention are described in moredetail below.

4. DETAILED DESCRIPTION OF THE INVENTION

In the description that follows in section 4, reference to variousproteins, isoforms thereof and treatment of diseases/disorders are inrelation to human proteins, isoforms thereof and human diseases anddisorders. Thus “NRG-1”, “NRG1a” and “NRG1β1” are to be construed asreferring to human NRG-1 etc. In the embodiments described below,reference to “NRG1” and “NRG1β1” may be taken to refer to all forms ofthe respective protein and additionally and individually, to soluble andmembrane bound forms and the reader of this specification may assumethat each embodiment is intended to be construed as such. Therefore,unless specified otherwise, in each embodiment described below refers tothree embodiments, firstly to all forms of the respective protein,secondly to any membrane bound form and thirdly to soluble forms of therespective protein.

4.1 Binding Compounds

In one aspect of the present invention, there is provided a bindingcompound that neutralizes the biological activity of a NRG1 protein.

In some embodiments, the binding compound binds with and neutralizes theability of NRG1β1 to promote expression of MUC5AC and MUC5B onepithelial cells (e.g. goblet cells). Such binding compounds may bindwith and inhibit NRG1β1 binding with its cognate receptor e.g. ErbB2and/or ErbB3, preferably the ErbB2/ErbB3 heterodimer. In suchembodiments, the binding compound maybe a low molecular weight chemicalentity capable of binding with a NRG-1 protein such as NRG-1β1 andthereby neutralize its biological activity by e.g. inhibiting theinteraction between the protein and its cognate receptor, e.g. ErbB2and/or ErbB3. In other such embodiments, the binding compound maybe atherapeutic protein such as an antibody capable of binding with andinhibiting the interaction between the NRG-1 protein (e.g. NRG-1β1) andits cognate receptor (e.g. ErbB2 and/or ErbB3). These embodiments aredescribed in more detail below. In another embodiment, the bindingcompound may bind with ADAM-17 and inhibit the activity thereof, to, inturn inhibit the formation of soluble NRG1β1 from its membrane boundprecursor form.

In another embodiment, the binding compound may inhibit the expressionof NRG-1, e.g. NRG-1β1 and thereby neutralize the ability of NRG1 topromote MUC5AC and MUC5B protein expression on epithelial cells. Inthese embodiments, the binding compound may inhibit the expression ofNRG-1 (e.g. NRG1β1) at the level of transcription and/or translation.For example, the binding compound maybe an anti-sense oligonucleotidecapable of binding with the complementary region of the NRG-1 gene andthereby inhibit its transcription. In other such embodiments, thebinding compound maybe a short interfering RNA (siRNA) capable ofinhibiting the translation of a RNA compound capable of encoding a NRG-1(e.g. NRG1β1) protein. These embodiments are described in some moredetail below.

4.1—Therapeutic Proteins

A therapeutic protein of the present invention maybe an antibody,Adnectin, Ankyrin, Maxybody/Avimer, Affibody, anticalin, or Affilin.

4.1.1.—Antibodies

Antibodies of the present invention maybe in any of a number of formatswell known to the skilled person. These formats include intactantibodies, various antibody fragments and other engineered formats asdescribed below. In preferred forms, antibodies of the present inventionare provided as a monoclonal population.

4.1.1.1—Intact Antibodies

Intact antibodies include heteromultimeric glycoproteins comprising atleast two heavy and two light chains. Aside from IgM, intact antibodiesare usually heterotetrameric glycoproteins of approximately 150 KDa,composed of two identical light (L) chains and two identical heavy (H)chains. Typically, each light chain is linked to a heavy chain by onecovalent disulfide bond while the number of disulfide linkages betweenthe heavy chains of different immunoglobulin isotypes varies. Each heavyand light chain also has intrachain disulfide bridges. Each heavy chainhas at one end a variable domain (VH) followed by a number of constantregions. Each light chain has a variable domain (VL) and a constantregion at its other end; the constant region of the light chain isaligned with the first constant region of the heavy chain and the lightchain variable domain is aligned with the variable domain of the heavychain. The light chains of antibodies from most vertebrate species canbe assigned to one of two type called Kappa or Lambda based on the aminoacid sequence of the constant region. Depending on the amino acidsequence of the constant region of their heavy chains, human antibodiescan be assigned to five different classes, IgA, IgD, IgE, IgG and IgM.IgG and IgA can be further subdivided into subclasses, IgG1, IgG2, IgG3and IgG4; and IgA1 and IgA2. Species variants exist with mouse and rathaving at least IgG2a, IgG2b. The variable domain of the antibodyconfers binding specificity upon the antibody with certain regionsdisplaying particular variability called complementarity determiningregions (CDRs). The more conserved portions of the variable region arecalled Framework regions (FR). The variable domains of intact heavy andlight chains each comprise four FR connected by three CDRs. The CDRs ineach chain are held together in close proximity by the FR regions andwith the CDRs from other chain contribute to the formation of theantigen binding site of antibodies. The constant regions are notdirectly involved in the binding of the antibody to the antigen butexhibit various effector functions such as participation in antibodydependent cell-mediated cytoxicity (ADCC), phagocytosis via binding toFcγr receptor, half-life/clearance rate via neonatal Fc receptor (FcRn)and complement dependent cytoxicity via the Clq component of thecomplement cascade.

Thus in one embodiment of the invention there is provided an intacttherapeutic antibody capable of binding NRG1 (e.g. NRG1β1) andneutralizing the biological activity thereof. In particular the intacttherapeutic antibody binds with NRG1 (e.g. NRG1β1 and inhibits theinteraction between NRG1 (or NRG1β1) and its cognate receptor ErbB2and/or ErbB3, particularly the ErbB2/ErbB3 heterodimer. In typicalembodiments, the antibody comprises a primate, and in particular a humanconstant region of an IgG isotype, e.g. IgG1 or IgG4 and is a human,humanised or chimeric antibody as described below

In another embodiment, there is provided an intact therapeutic antibodycapable of preferentially binding NRG1β1 (compared with NRG1a) andneutralizing the biological activity thereof. In particular the intacttherapeutic antibody preferentially binds with NRG1β1 and inhibits theinteraction between NRG1β1 and its cognate receptor ErbB2 and/or ErbB3,particularly the ErbB2/ErbB3 heterodimer. In preferred forms, the intacttherapeutic antibody comprises a primate, and in particular a humanconstant region of an IgG isotype, e.g. IgG1 or IgG4 and is a human,humanised or chimeric antibody as described below. The term“preferentially binds” and grammatical variations thereof usedthroughout this specification refers to the ability of the therapeuticprotein (e.g. antibody) to bind NRG1β1 with a higher affinity (at least2 fold) than it binds NRG1 a. The preferentially binding protein howeveris capable of neutralising to some significant degree the same sharedbiological activity of both NRG1β1 and NRG1a.

In another embodiment, there is provided an intact therapeutic antibodycapable of specifically binding NRG1β1 (compared with NRG1a) andneutralizing the biological activity thereof. In particular the intacttherapeutic antibody specifically binds with NRG1β1 and inhibits theinteraction between NRG1β1 and its cognate receptor ErbB2 and/or ErbB3,particularly the ErbB2/ErbB3 heterodimer. In preferred forms, the intacttherapeutic antibody comprises a primate, and in particular a humanconstant region of an IgG isotype, e.g. IgG1 or IgG4 and is a human,humanised or chimeric antibody as described below. The term“specifically binds” and grammatical variations thereof used throughoutthe present specification refers to the ability of the therapeuticprotein (e.g. antibody) to bind with NRG1β1 with a higher (e.g. at least5 fold higher) binding affinity than it does to NRG1a. The specificallybinding therapeutic protein is capable of neutralising NRG1β1 biologicalactivity but does not, to any significant degree, neutralise the sameshared biological activity of NRG1a.

In one embodiment of the invention there is provided an intacttherapeutic antibody capable of binding membrane bound NRG1 (e.g.NRG1β1) and neutralizing the biological activity thereof. In particularthe intact therapeutic antibody binds with membrane NRG1 (e.g. NRG1β1and inhibits the interaction between NRG1 (or NRG1β1) and its cognatereceptor ErbB2 and/or ErbB3, particularly the ErbB2/ErbB3 heterodimer.The intact therapeutic antibody may, for example, bind with membranebound NRG1 (e.g. NRG1β1) and inhibit formation of soluble NRG1 (e.g.NRG1β1) therefrom by, e.g. inhibiting cleavage of membrane bound NRG1(e.g. NRG1β1) or by promoting recycling of membrane NRG1 (e.g. NRG1β1).In typical embodiments, the antibody comprises a primate, and inparticular a human constant region of an IgG isotype, e.g. IgG1 or IgG4and is a human, humanised or chimeric antibody.

In another embodiment of the invention there is provided an intacttherapeutic antibody capable of binding soluble NRG1 (e.g. NRG1β1) andneutralizing the biological activity thereof. In particular the intacttherapeutic antibody binds with soluble NRG1 (e.g. NRG1β1 and inhibitsthe interaction between NRG1 (or NRG1β1) and its cognate receptor ErbB2and/or ErbB3, particularly the ErbB2/ErbB3 heterodimer. In typicalembodiments, the antibody comprises a primate, and in particular a humanconstant region of an IgG isotype, e.g. IgG1 or IgG4 and is a human,humanised or chimeric antibody.

4.1.1.1.1 Human Antibodies

Human antibodies may be produced by a number of methods known to thoseof skill in the art. Human antibodies can be made by the hybridomamethod using human myeloma or mouse-human heteromyeloma cells lines seeKozbor J. Immunol. 133, 3001, (1984) and Brodeur, Monoclonal AntibodyProduction Techniques and Applications, pp 51-63 (Marcel Dekker Inc,1987). Alternative methods include the use of phage libraries ortransgenic mice both of which utilize human V region repertories (seeWinter G, (1994), Annu. Rev. Immunol. 12, 433-455, Green L L (1999), J.Immunol. Methods 231, 11-23).

Several strains of transgenic mice are now available wherein their mouseimmunoglobulin loci has been replaced with human immunoglobulin genesegments (see Tomizuka K, (2000) PNAS 97, 722-727; Fishwild D M (1996)Nature Biotechnol. 14, 845-851. Mendez M J, 1997, Nature Genetics, 15,146-156). Upon antigen challenge such mice are capable of producing arepertoire of human antibodies from which antibodies of interest can beselected. Of particular note is the Trimera™ system (see Eren R et al,(1988) Immunology 93:154-161) where human lymphocytes are transplantedinto irradiated mice, the Selected Lymphocyte Antibody System (SLAM, seeBabcook et al, PNAS (1996) 93: 7843-7848) where human (or other species)lymphocytes are effectively put through a massive pooled in vitroantibody generation procedure followed by deconvulated, limitingdilution and selection procedure and the Xenomouse™ (Abgenix Inc). Analternative approach is available from Morphotek Inc using theMorphodoma™ technology. Phage display technology can be used to producehuman antibodies (and fragments thereof), see McCafferty; Nature, 348,552-553 (1990) and Griffiths A D et al (1994) EMBO 13: 3245-3260.According to this technique antibody V domain genes are cloned in frameinto either a major or minor coat of protein gene of a filamentousbacteriophage such as M13 or fd and displayed (usually with the aid of ahelper phage) as function antibody fragments on the surface of the phageparticle. Selections based on the function properties of the antibodyresult in selection of the gene encoding the antibody exhibiting theseproperties. The phage display technique can be used to select antigenspecific antibodies from libraries made from human B cells taken fromindividuals afflicted with a disease or disorder described above oralternatively from unimmunized human donors (see Marks; J Mol Bio 222,581-591, 1991). Where an intact human antibody is desired comprising anFc domain it is necessary redone the phage displayed derived fragmentinto a mammalian expression vectors comprising the desired constantregions and establishing stable expressing cell lines.

The technique of affinity maturation (Marks; Bio/technol 10, 779-783(1992)) may be used to provide binding affinity wherein the affinity ofthe primary human antibody is improved by sequentially replacing the Hand L chain V regions with naturally occurring variants and selecting onthe basis of improved binding affinities. Variants of this techniquesuch as ‘epitope imprinting’ are now also available, see WO 93/06213.See also Waterhouse; Nucl Acids Res 21, 2265-2266 (1993).

Thus in one embodiment of the invention there is provided an intacttherapeutic human antibody capable of binding NRG1 (e.g. NRG1β1) andneutralizing the biological activity thereof. In particular the intacttherapeutic human antibody binds with NRG 1β1 and inhibits theinteraction between NRG1β1 and its cognate receptor ErbB2 and/or ErbB3,particularly the ErbB2/ErbB3 heterodimer. In typical embodiments theintact therapeutic human antibody comprises a constant region of an IgGisotype, e.g. IgG1 or IgG4.

In another embodiment, there is provided an intact therapeutic humanantibody capable of preferentially binding NRG1β1 (compared with NRG1a)and neutralizing the biological activity thereof. In particular theintact therapeutic human antibody preferentially binds with NRG1β1 andinhibits the interaction between NRG1 and its cognate receptor ErbB2and/or ErbB3, particularly ErbB2/ErbB3 heterodimer. In preferred forms,the intact therapeutic human antibody comprises a constant region of anIgG isotype, e.g. IgG1 or IgG4.

In another embodiment, there is provided an intact therapeutic humanantibody capable of specifically binding NRG1β1 (compared with NRG1a)and neutralizing the biological activity thereof. In particular theintact therapeutic human antibody specifically binds with NRG1β1 andinhibits the interaction between NRG1β1 and its cognate receptor ErbB2and/or ErbB3. In preferred forms, the intact therapeutic human antibodycomprises a constant region of an IgG isotype, e.g. IgG1 or IgG4.

4.1.1.1.2 Chimaeric and Humanised Antibodies

The use of intact non-human antibodies in the treatment of humandiseases or disorders carries with it the potential for the now wellestablished problems of immunogenicity, that is the immune system of thepatient may recognises the non-human intact antibody as non-self andmount a neutralising response. This is particularly evident uponmultiple administration of the non-human antibody to a human patient.Various techniques have been developed over the years to overcome theseproblems and generally involve reducing the composition of non-humanamino acid sequences in the intact antibody whilst retaining therelative ease in obtaining non-human antibodies from an immunisedanimal, e.g. mouse, rat or rabbit. Broadly two approaches have been usedto achieve this. The first are chimaeric antibodies, which generallycomprise a non-human (e.g. rodent such as mouse) variable domain fusedto a human constant region. Because the antigen-binding site of anantibody is localised within the variable regions the chimaeric antibodyretains its binding affinity for the antigen but acquires the effectorfunctions of the human constant region and are therefore able to performeffector functions such as described supra. Chimaeric antibodies aretypically produced using recombinant DNA methods. DNA encoding theantibodies (e.g. cDNA) is isolated and sequenced using conventionalprocedures (e.g. by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the H and L chains of theantibody of the invention, e.g. DNA encoding SEQ ID NO 1, 2, 3, 4, 5 and6 described supra). Hybridoma cells serve as a typical source of suchDNA. Once isolated, the DNA is placed into expression vectors which arethen transfected into host cells such as E. Coli, COS cells, CHO cellsor myeloma cells that do not otherwise produce immunoglobulin protein toobtain synthesis of the antibody. The DNA may be modified bysubstituting the coding sequence for human L and H chains for thecorresponding non-human (e.g. murine) H and L constant regions see e.g.Morrison; PNAS 81, 6851 (1984).

The second approach involves the generation of humanised antibodieswherein the non-human content of the antibody is reduced by humanizingthe variable regions. Two techniques for humanisation have gainedpopularity. The first is humanisation by CDR grafting. CDRs build loopsclose to the antibody's N-terminus where they form a surface mounted ina scaffold provided by the framework region. Antigen-binding specificityof the antibody is mainly defined by the topography and by the chemicalcharacteristics of its CDR surface. These features are in turndetermined by the conformation of the individual CDRs, by the relativedisposition of the CDRs, and by the nature and disposition of the sidechains of the residues comprising the CDRs. A large decrease inimmunogenicity can be achieved by grafting only the CDRs of a non-human(e.g. murine) antibodies (‘donor’ antibodies) onto human framework(‘acceptor framework’) and constant regions (see Jones et al (1986)Nature 321, 522-525 and Verhoeyen M et al (1988) Science 239,1534-1536). However, CDR grafting per se may not result in the completeretention of antigen-binding properties and it is frequency found thatsome framework residues (sometimes referred to as ‘backmutations’) ofthe donor antibody need to be preserved in the humanised compound ifsignificant antigen-binding affinity is to be recovered (see Queen C etal (1989) PNAS 86, 10, 029-10,033, Co, M et al (1991) Nature 351,501-502). In this case, human V regions showing the greatest sequencehomology to the non-human donor antibody are chosen from a database inorder to provide the human framework (FR). The selection of human FRscan be made either from human consensus or individual human antibodies.Where necessary key residues from the donor antibody are substitutedinto the human acceptor framework to preserve CDR conformations.Computer modelling of the antibody may be used to help identify suchstructurally important residues, see WO99/48523.

Alternatively, humanisation may be achieved by a process of ‘veneering’.A statistical analysis of unique human and murine immunoglobulin heavyand light chain variable regions revealed that the precise patterns ofexposed residues are different in human and murine antibodies, and mostindividual surface positions have a strong preference for a small numberof different residues (see Padlan E A, et al; (1991) Mol Immunol 28,489-498 and Pedersen J T et al (1994) J Mol Biol 235; 959-973).Therefore it is possible to reduce the immunogenicity of a non-human Fvby replacing exposed residues in its framework regions that differ fromthose usually found in human antibodies. Because protein antigenicitymay be correlated with surface accessibility, replacement of the surfaceresidues may be sufficient to render the mouse variable region‘invisible’ to the human immune system (see also Mark G E et al (1994)in Handbook of Experimental Pharmacology vol 113: The pharmacology ofmonoclonal Antibodies, Springer-Verlag, pp 105-134). This procedure ofhumanisation is referred to as ‘veneering’ because only the surface ofthe antibody is altered, the supporting residues remain undisturbed.

Thus in one embodiment of the invention there is provided an intacttherapeutic humanised antibody capable of binding NRG1 (e.g. NRG1β1) andneutralizing the biological activity thereof. In particular the intacttherapeutic humanised antibody binds with NRG1β1 and inhibits theinteraction between NRG1β1 and its cognate receptor ErbB2 and/or ErbB3.In typical embodiments the intact therapeutic humanised antibodycomprises a constant region of an IgG isotype, e.g. IgG1 or IgG4.

In another embodiment, there is provided an intact therapeutic humanisedantibody capable of preferentially binding NRG1β1 (compared with NRG1a)and neutralizing the biological activity thereof. In particular theintact therapeutic humanised antibody preferentially binds with NRG1β1and inhibits the interaction between NRG1β1 and its cognate receptorErbB2 and/or ErbB3. In preferred forms, the intact therapeutic humanantibody comprises a constant region of an IgG isotype, e.g. IgG1 orIgG4.

In another embodiment, there is provided an intact therapeutic humanisedantibody capable of specifically binding NRG1β1 (compared with NRG1a)and neutralizing the biological activity thereof. In particular theintact therapeutic humanised antibody specifically binds with NRG1β1 andinhibits the interaction between NRG1 and its cognate receptor ErbB2and/or ErbB3. In preferred forms, the intact therapeutic humanisedantibody comprises a primate, and in particular a human constant regionof an IgG isotype, e.g. IgG1 or IgG4.

4.1.1.1.3 Bispecific Antibodies

A bispecific antibody is an antibody having binding specificities for atleast two different epitopes. Methods of making such antibodies areknown in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the coexpression of two immunoglobulinH chain-L chain pairs, where the two H chains have different bindingspecificities, (see Millstein et al, Nature 305, 537-539 (1983),WO93/08829 and Traunecker et al, EMBO, 10, 1991, 3655-3659). Because ofthe random assortment of H and L chains, a potential mixture of tendifferent antibody structures are produced of which only one has thedesired binding specificity. An alternative approach involves fusing thevariable domains with the desired binding specificities to heavy chainconstant region comprising at least part of the hinge region, CH2 andCH3 regions. It is preferred to have the CH1 region containing the sitenecessary for light chain binding present in at least one of thefusions. DNA encoding these fusions and, if desired, the L chain areinserted into separate expression vectors and are the contransfectedinto a suitable host organism. It is possible though to insert thecoding sequences for two or all three chains into one expression vector.In one preferred approach, the bispecific antibody is composed of an Hchain with a first binding specificity in one arm and an H-L chain pair,providing a second binding specificity in the other arm, see WO94/04690.Also see Suresh et al, Methods in Enzymology 121, 210, 1986.

In one embodiment of the invention there is provided a bispecifictherapeutic antibody wherein at least one binding specificity of saidantibody is for NRG1, particularly NRG1β1, wherein said antibody bindswith and neutralises NRG1 (e.g. NRG1β1) biological activity. Inpreferred forms the bispecific antibody comprises a primate, e.g. humanantibody of a IgG (e.g. IgG1 or IgG4) isotype.

4.1.1.1.4 Antibody Fragments

In certain embodiments of the invention there is provided therapeuticantibody fragments which modulate (e.g. inhibit) the interaction betweenNRG1 and its cognate receptor e.g. ErbB2 and/or ErbB3 for exampleErbB2/ErbB3 heterodimer. Such fragments may be functional antigenbinding fragments of intact and/or humanised chimaeric antibodies suchas Fab, Fab′, F(ab′)₂, Fv, ScFv fragments of the antibodies describedsupra.

Traditionally, such fragments are produced by the proteolytic digestionof intact antibodies by e.g. papain digestion (see for example WO94/29348) but may be produced directly from recombinantly transformedhost cells. For the production of ScFv, see Bird et al; (1988) Science,242, 423-426. In addition, antibody fragments may be produced using avariety of engineering techniques as described below.

FV fragments appear to have lower interaction energy of their two chainsthan Fab fragments. To stabilise the association of the VH and VLdomains, they have been linked with peptides (Bird et al, (1988)Science, 242, 423-426, Huston et al, PNAS, 85, 5879-5883), disulphidebridges (Glockshuber et al, (1990) Biochemistry, 29, 1362-1367) and‘knob in hole’ mutations (Zhu et al (1997), Protein Sci., 6, 781-788).ScFv fragments can be produced by methods well known to those skilled inthe art (see Whitlow et al (1991), Methods companion Methods Enzymol, 2,97-105 and Huston et al (1993) Int Rev Immunol 10, 195-217. ScFv may beproduced in bacterial cells such as E. Coli but are more preferablyproduced in eukaryotic cells. One disadvantage of ScFv is themonovalency of the product, which precludes an increased avidity due topolyvalent binding, and their short half-life. Attempts to overcomethese problems include bivalent (ScFv′)₂ produced from ScFv containingan additional C terminal cysteine by chemical coupling (Adams et al(1993) Can Res 53, 4026-4034 and McCartney et al (1995) Protein Eng, 8,301-314) or by spontaneous site-specific dimerization of ScFv containingan unpaired C terminal cysteine residue (see Kipriyanov et al (1995)Cell. Biophys 26, 187-204). Alternatively, ScFv can be forced to formmultimers by shortening the peptide linker to 3 and 12 residues to form‘diabodies’ (see Holliger et al PNAS (1993), 90, 6444-6448). Reducingthe linker still further can result in ScFV trimers (‘triabodies’, seeKortt et al (1997) Protein Eng, 10, 423-433) and tetramers(‘tetrabodies’, see Le Gall et al (1999) FEBS Lett, 453, 164-168).Construction of bialent ScFV compounds can also be achieved by geneticfusion with protein dimerzing motifs to form ‘miniantibodies’ (see Packet al (1992) Biochemistry 31, 1579-1584) and ‘minibodies’ (see Hu et al(1996), Cancer Res. 56, 3055-3061). ScFv-Sc-Fv tandems ((ScFV)₂) mayalso be produced by linking two ScFV units by a third peptide linger,(see Kurucz et al (1995) J Immunol, 154, 4576-4582). Bispecificdiabodies can be produced through the noncovalent association of twosingle chain fusion products consisting of VH domain from one antibodyconnected by a short linker to the VL domain of another antibody, (seeKipriyanov et al (1998), Int J Can 77, 763-772). The stability of suchbispecific diabodies can be enhanced by the introduction of disulphidebridges or ‘knob in hole’ mutations as described supra or by theformation of single chain diabodies (ScDb) wherein two hydrid ScFvfragments are connected through a peptide linker (see Kontermann et al(1999) J Immunol Methods 226, 179-188). Tetravalent bispecific compoundsare available by e.g. fusing a ScFv fragment to the CH3 domain of an IgGcompound or to a Fab fragment through the hinge region (see Coloma et al(1997) Nature Biotechnol, 15, 159-163). Alternatively, tetravalentbispecific compounds have been created by the fusion of bispecificsingle chain diabodies (see Alt et al (1999) FEBS Lett 454, 90-94).Smaller tetravalent bispecific compounds can also be formed by thedimerization of either ScFv-ScFv tandems with a linker containing ahelix-loop-helix motif (DiBi miniantibodies, see Muller et at (1998)FEBS Lett 432, 45-49) or a single chain compound comprising fourantibody variable domains (VH and VL) in an orientation preventingintramolecular pairing (tandem diabody, see Kipriyanov et al, (1999) JMol Biol 293, 41-56). Bispecific F9ab′)₂ fragments can be created bychemical coupling of Fab′ fragments or by heterodimerization throughleucine zippers (see Shalaby et al (1992) J Exp Med 175, 217-225 andKostelny et al (1992), J Immunol 148 1547-1553). Also available areisolated VII and VL domains (Domantis plc), see U.S. Pat. No. 6,248,516;U.S. Pat. No. 6,291,158; U.S. Pat. No. 6,172,197 and isolated VHH domainantibodies (Nanobodies). These domain and nanobodies may be dualspecific having one specificity directed to a half life extendingprotein such as human serum albumin (HSA). Such domain and nanobodiesboth monospecific for a NRG1 protein of the invention and further dualspecific for a half life extending protein such as HSA are specificallycontemplated by the invention.

In one embodiment there is provided a therapeutic antibody fragment(e.g. ScFv, Fab, Fab′, F(ab′)₂) or an engineered antibody fragment asdescribed supra that binds (e.g. preferentially or specifically binds)with NRG1, e.g. NRG1β1 and neutralises the biological activity thereofby e.g. inhibiting the interaction between NRG1 and its cognate receptore.g. ErbB2 and/or ErbB3, for example ErbB2/ErbB3 heterodimer.

4.1.1.1.5 Heteroconjugate Antibodies

Heteroconjugate antibodies also form an embodiment of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies formed using any convenient cross-linking methods.See, for example, U.S. Pat. No. 4,676,980.

4.1.1.1.6 Other Modifications

The interaction between the Fc region of an antibody and various Fcreceptors (FcγR) is believed to mediate the effector functions of theantibody which include antibody-dependent cellular cytotoxicity (ADCC),fixation of complement, phagocytosis, and half-life/clearance of theantibody. Various modifications to the Fc region of antibodies of theinvention may be carried out depending on the desired property. Forexample, specific mutations in the Fc region to render an otherwiselytic antibody, non-lytic is detailed in EP 0629 240B1 and Ep 0307 434B2or one may incorporate a salvage receptor binding epitope into theantibody to increase serum half-life, see U.S. Pat. No. 5,739,277. Thereare five currently recognised human Fcγ, FcγR (I), FCγRIIb, FcγRIIIa andneonatal FcRn. Shields et al, (2001) J Biol Chem 276, 6591-6604demonstrated that a common set of IgG1 residues is involved in bindingall FcγRs, while FCγRII and FcγRIII utilize distinct sites outside ofthis common set. One group of IgG1 residues reduced binding to all FcγRswhen altered to alanine: Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239.All are in the IgG CH2 domain and clustered near the hinge joining CH1and CH2. While FcγRI utilizes only the common set of IgG1 residues forbinding, FcγRII and FcγRIII (e.g. Glu-293). Some variants showedimproved binding to FcγRII or FcγRIII but did not affect binding to theother receptor (e.g. Ser-267Ala improved binding to FcγRII but bindingto FcγRIII was unaffected).

Other variants exhibited improved binding to DcyRII or FcγRIII withreduction in binding to the other receptor (e.g. Ser298Ala improvedbinding to FcγRIII and reduced binding to FcγRII). For FcγRIIIa, thebest binding IgG1 variants had combined alanine substitutions atSer-298, Glu-333 and Ls-334. The neonatal FcRn receptor is believed tobe involved in both antibody clearance and the transcytosis acrosstissues (see Junghans R P (1997) Immunol Res 16, 2957 and Ghetie et al(2000) Annu Rev Immunol 18, 739-766). Human IgG1 residues determined tointeract directly with human FcRn included Ile253, Ser254, Lys288,Thr307, Gln311, Asn434 and His435. Switches at any of these positionsdescribed in this section may enable increased serum half-life and/oraltered effector properties of antibodies of the invention and thereforeforms an embodiment of the invention.

Other modifications include glycosylation variants of the antibodies ofthe invention. Glycosylation of antibodies at conserved positions intheir constant regions is known to have a profound effect on antibodyfunction, particularly effector functioning such as those describedabove, see for example, Boyd et al (1996), Mol Immunol 32, 1311-1318.Glycosylation variants of the therapeutic antibodies or antigen bindingfragments thereof of the present invention wherein one or morecarbohydrate moiety is added, substituted, deleted or modified arecontemplated. Introduction of an asparagine-X-serin orasparagine-X-threonine motif creates a potential side for enzymaticattachment of carbohydrate moieties and may therefore be used tomanipulate the glycosylation of an antibody. In Raju et al (2001)Biochemistry 40, 8868-8876 the terminal sialyation of a TNFR-IgGimmunoadhesin was increased through a process of regalactosylationand/or resialylation using beta-1,4-galactrosyltransferace and/or alpha,2,3 sialyltransferase. Increasing the terminal sialylation is believedto increase the half-life of the immunoglobulin. Antibodies, in commonwith most glycoproteins, are typically produced as a mixture ofglycoforms. This mixture is particularly apparent when antibodies areproduced in eukaryotic, particularly mammalian cells. A variety ofmethods have been developed to manufacture defined glycoforms, see Zhanget al, Science (2004), 303, 371; Sears et al, Science (2001), 291, 2344;Wacker et al (2002), Science 298, 1790; Davis et al (2002), Chem Rev102, 579; Hang et al (2001), Acc Chem Res 34, 727. Thus the inventioncontemplates a plurality of therapeutic (monoclonal) antibodies (whichmay be of the IgG isotype, e,g. IgG1) as herein described comprising adefined number (e.g. 7 or less, for example 5 or less such as two or asingle) glycoform(s) or said antibodies or antigen binding fragmentsthereof.

Further embodiments of the invention include therapeutic antibodies ofthe invention or antigen binding fragments thereof coupled to anon0proteinaeous polymer such as polyethylene glycol (PEG),polypropylene glycol or polyoxyalkylene. Conjugation of proteins to PEGis an established technique for increasing half-life of proteins, aswell as reducing antigenicity and immunogenicity of proteins. The use ofPEGylation with different molecular weights and styles (linear orbranched) has been investigated with intact antibodies as well as Fab′fragments (see Koumenis I L et al (2000) Int I Pharmaceut 198; 83-95.

4.2 Adnectins—Compound Therapeutics

The adnectin scaffolds are based on fibronectin type III domain (e.g.,the tenth module of the fibronectin type III (10 Fn3 domain). Thefibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands. (U.S. Pat. No.6,818,418).

These fibronectin-based scaffolds are not an immunoglobulin, althoughthe overall fold is closely related to that of the smallest functionalantibody fragment, the variable region of the heavy chain, whichcomprises the entire antigen recognition unit in camel and llama IgG.Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedcompounds can be used as scaffolds where the loop regions of thecompound can be replaced with CDRs of the invention using standardcloning techniques. Accordingly, in some embodiments there is providedan adnectin compound that binds with and neutralises the biologicalactivity of NRG1 and in particular NRG1β1.

4.3 Ankyrin—Molecular Partners

This technology is based on using proteins with ankyrin derived repeatmodules as scaffolds for bearing variable regions which can be used forbinding to different targets. The ankyrin repeat module is a 33 aminoacid polypeptide consisting of two anti-parallel a-helices and a β-turn.Binding of the variable regions is mostly optimized by using ribosomedisplay. Accordingly, in some embodiments there is provided an Ankyrincompound that binds with and neutralises the biological activity of NRG1and in particular NRG1β1.

4.4 Maxybodies/Avimers—Avidia

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example,US20040175756; US20050053973; US20050048512; and US20060008844.Accordingly, in some embodiments there is provided an Maxybody compoundthat binds with and neutralises the biological activity of NRG1 and inparticular NRG1β1.

4.5 Protein A—Affibody

Affibody® affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate Affibody® libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibody®compounds mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of Affibody® compounds issimilar to that of an antibody. Accordingly, in some embodiments thereis provided an Protein A-affibody compound that binds with andneutralises the biological activity of NRG1 and in particular NRG1β1.

4.6 Anticalins—Pieris

Anticalins® are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compound s. Several naturallipocalins occur in human tissues or body liquids.

The protein architecture is reminiscent of immunoglobulins, withhypervariable loops on top of a rigid framework. However, in contrastwith antibodies or their recombinant fragments, lipocalins are composedof a single polypeptide chain with 160 to 180 amino acid residues, beingjust marginally bigger than a single immunoglobulin domain.

The set of four loops, which makes up the binding pocket, showspronounced structural plasticity and tolerates a variety of side chains.The binding site can thus be reshaped in a proprietary process in orderto recognize prescribed target compounds of different shape with highaffinity and specificity.

One protein of lipocalin family, the bilin-binding protein (BBP) ofPieris Brassicae has been used to develop anticalins by mutagenizing theset of four loops. One example of a patent application describing“anticalins” is PCT WO 199916873.

Accordingly, in some embodiments there is provided an anticalin compoundthat binds with and neutralises the biological activity of NRG1 and inparticular NRG1β1.

4.7 Affilin—Scil Proteins

Affilin™ compounds are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small compounds.New Affilin™ compounds can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.Affilin™ compounds do not show any structural homology to immunoglobulinproteins. Scil Proteins employs two Affilin™ scaffolds, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368. Accordingly, insome embodiments there is provided an Affilin compound that binds withand neutralises the biological activity of NRG1 and in particularNRG1β1.

4.7.1—Other Therapeutic Modalities

As noted previously, other therapeutic modalities of this inventioninclude modulators (particularly inhibitors) of NRG1, and in particularNRG1β1 which exert their effect on their target prior to proteinexpression. Examples include anti-sense oligonucleotides that comprise(or consist essentially of) a sequence (a) capable of forming a stabletriplex with a portion of the NRG1 (particularly NRG1β1) gene, or (b)capable of forming a stable duplex with a portion of an mRNA transcriptof the NRG1 (particularly NRG1β1) gene under physiological conditions.Other examples include molecules that can participate in the phenomenaof “RNA interference”. RNA interference (RNAi) is particularly usefulfor specifically inhibiting the production of a particular protein.Although not wishing to be limited by theory, Waterhouse et al. (1998)have provided a model for the mechanism by which dsRNA (duplex RNA) canbe used to reduce protein production. This technology relies on thepresence of dsRNA molecules that contain a sequence that is essentiallyidentical to the mRNA of the gene of interest or part thereof.Conveniently, the dsRNA can be produced from a single promoter in arecombinant vector or host cell, where the sense and anti-sensesequences are flanked by an unrelated sequence which enables the senseand anti-sense sequences to hybridise to form the dsRNA molecule withthe unrelated sequence forming a loop structure. The design andproduction of suitable dsRNA molecules for the present invention is wellwithin the capacity of a person skilled in the art, particularlyconsidering Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619,WO 99/53050, WO 99/49029, and WO 01/34815.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded RNA product(s) with homology to the targetgene to be inactivated.

The DNA therefore comprises both sense and antisense sequences that,when transcribed into RNA, can hybridise to form the double-stranded RNAregion. In a preferred embodiment, the sense and antisense sequences areseparated by a spacer region that comprises an intron which, whentranscribed into RNA, is spliced out. This arrangement has been shown toresult in a higher efficiency of gene silencing. The double-strandedregion may comprise one or two RNA molecules, transcribed from eitherone DNA region or two. The presence of the double stranded molecule isthought to trigger a response from an endogenous mammalian system thatdestroys both the double stranded RNA and also the homologous RNAtranscript from the target mammalian gene, efficiently reducing oreliminating the activity of the target gene.

The length of the sense and antisense sequences that hybridise shouldeach be at least 19 contiguous nucleotides, preferably at least 30 or 50nucleotides, and more preferably at least 100, 200, 500 or 1000nucleotides. The full-length sequence corresponding to the entire genetranscript may be used. The lengths are most preferably 100-2000nucleotides. The degree of identity of the sense and antisense sequencesto the targeted transcript should be at least 85%, preferably at least90% and more preferably 95-100%. The RNA molecule may of course compriseunrelated sequences which may function to stabilise the molecule. TheRNA molecule may be expressed under the control of a RNA polymerase IIor RNA polymerase HI promoter. Examples of the latter include tRNA orsnRNA promoters.

Preferred small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-21 contiguousnucleotides of the target mRNA. Preferably, the siRNA sequence commenceswith the dinucleotide AA, comprises a GC-content of about 30-70%(preferably, 30-60%, more preferably 40-60% and more preferably about45%-55%), and does not have a high percentage identity to any nucleotidesequence other than the target in the genome of the mammal in which itis to be introduced, for example as determined by standard BLAST search.MicroRNA regulation is a clearly specialised branch of the RNA silencingpathway that evolved towards gene regulation, diverging fromconventional RNAi/PTGS.

MicroRNAs are a specific class of small RNAs that are encoded ingene-like elements organised in a characteristic inverted repeat. Whentranscribed, microRNA genes give rise to stem-looped precursor RNAs fromwhich the microRNAs are subsequently processed. MicroRNAs are typicallyabout 21 nucleotides in length. The released miRNAs are incorporatedinto RISC-like complexes containing a particular subset of Argonauteproteins that exert sequence-specific gene repression (see, for example,Millar and Waterhouse, 2005; Pasquinelli et al. 2005; Almeida andAllshire, 2005).

4.8 Production Methods

Therapeutic proteins of the invention, and particularly antibodies maybeproduced as a polyclonal population but are more preferably produced asa monoclonal population (that is as a substantially homogenouspopulation of identical antibodies directed against a specific antigenicbinding site). It will of course be apparent to those skilled in the artthat a population implies more than one antibody entity. Antibodies ofthe present invention may be produced in transgenic organisms such asgoats (see Pollock et al (1999), J. Immunol. Methods 231:147-157),chickens (see Morrow K J J (2000) Genet. Eng. News 20:1-55, mice (seePollock et an or plants (see Doran P M, (2000) Curr. Opinion Biotechnol.11, 199-204, Ma J K-C (1998), Nat. Med. 4; 601-606, Baez J e. al,BioPharm (2000) 13: 50-54, Stoger E et al; (2000) Plant Mol. Biol.42:583-590). Antibodies may also be produced by chemical synthesis.However, antibodies and other therapeutic proteins of the invention aretypically produced using recombinant cell culturing technology wellknown to those skilled in the art. A polynucleotide encoding theantibody is isolated and inserted into a replicable vector such as aplasmid for further cloning (amplification) or expression. One usefulexpression system is a glutamate synthetase system (such as sold byLonza Biologies), particularly where the host cell is CHO or NSO (seebelow). Polynucleotide encoding the antibody is readily isolated andsequenced using conventional procedures (e.g. oligonucleotide probes).Vectors that may be used include plasmid, virus, phage, transposons,minichromsomes of which plasmids are a typical embodiment. Generallysuch vectors further include a signal sequence, origin of replication,one or more marker genes, an enhancer element, a promoter andtranscription termination sequences operably linked to the light and/orheavy chain polynucleotide so as to facilitate expression.Polynucleotide encoding the light and heavy chains may be inserted intoseparate vectors and transfected into the same host cell or, if desiredboth the heavy chain and light chain can be inserted into the samevector for transfection into the host cell. Thus according to one aspectof the present invention there is provided a process of constructing avector encoding the light and/or heavy chains of a therapeutic antibodyor antigen binding fragment thereof of the invention, which methodcomprises inserting into a vector, a polynucleotide encoding either alight chain and/or heavy chain of a therapeutic antibody of theinvention.

4.8.1 Signal Sequences

Antibodies of the present invention maybe produced as a fusion proteinwith a heterologous signal sequence having a specific cleavage site atthe N terminus of the mature protein. The signal sequence should berecognised and processed by the host cell. For prokaryotic host cells,the signal sequence may be an alkaline phosphatase, penicillinase, orheat stable enterotoxin I1 leaders. For yeast secretion the signalsequences may be a yeast invertase leader, [alpha] factor leader or acidphosphatase leaders see e.g. WO90/13646. In mammalian cell systems,viral secretory leaders such as herpes simplex gD signal and a nativeimmunoglobulin signal sequence are available. Typically the signalsequence is ligated in reading frame to DNA encoding the antibody of theinvention.

4.8.2 Origin of Replication

Origin of replications are well known in the art with pBR322 suitablefor most gram-negative bacteria, 2μ plasmid for most yeast and variousviral origins such as SV40, polyoma, adenovirus, VSV or BPV for mostmammalian cells. Generally the origin of replication component is notneeded for mammalian expression vectors but the SV40 may be used sinceit contains the early promoter.

4.8.3 Selection Marker

Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins e.g. ampicillin, neomycin, methotrexate ortetracycline or (b) complement auxotrophic deficiencies or supplynutrients not available in the complex media. The selection scheme mayinvolve arresting growth of the host cell. Cells, which have beensuccessfully transformed with the genes encoding the therapeuticantibody of the present invention, survive due to e.g. drug resistanceconferred by the selection marker. Another example is the so-called DHFRselection marker wherein transformants are cultured in the presence ofmethotrexate. In typical embodiments, cells are cultured in the presenceof increasing amounts of methotrexate to amplify the copy number of theexogenous gene of interest. CHO cells are a particularly useful cellline for the DHFR selection. A further example is the glutamatesynthetase expression system (Lonza Biologies). A suitable selectiongene for use in yeast is the trp1 gene, see Stinchcomb et al Nature 282,38, 1979.

4.8.4 Promoters

Suitable promoters for expressing antibodies of the invention areoperably linked to DNA/polynucleotide encoding the antibody. Promotersfor prokaryotic hosts include phoA promoter, Beta-lactamase and lactosepromoter systems, alkaline phosphatase, tryptophan and hybrid promoterssuch as Tac. Promoters suitable for expression in yeast cells include3-phosphoglycerate kinase or other glycolytic enzymes e.g. enolase,glyceralderhyde 3 phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose 6 phosphate isomerase,3-phosphoglycerate mutase and glucokinase. Inducible yeast promotersinclude alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,metallothionein and enzymes responsible for nitrogen metabolism ormaltose/galactose utilization.

Promoters for expression in mammalian cell systems include viralpromoters such as polyoma, fowlpox and adenoviruses (e.g. adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus (inparticular the immediate early gene promoter), retrovirus, hepatitis Bvirus, actin, rous sarcoma virus (RSV) promoter and the early or lateSimian virus 40. Of course the choice of promoter is based upon suitablecompatibility with the host cell used for expression.

4.8.5 Enhancer Element

Where appropriate, e.g. for expression in higher eukaroytics, anenhancer element operably linked to the promoter element in a vector maybe used. Suitable mammalian enhancer sequences include enhancer elementsfrom globin, elastase, albumin, fetoprotein and insulin. Alternatively,one may use an enhancer element from a eukaroytic cell virus such asSV40 enhancer (at bp 100-270), cytomegalovirus early promoter enhancer,polyma enhancer, baculoviral enhancer or murine IgG2a locus (seeWO04/009823). The enhancer is preferably located on the vector at a siteupstream to the promoter.

4.8.6 Host Cells

Suitable host cells for cloning or expressing vectors encodingantibodies of the invention are prokaroytic, yeast or higher eukaryoticcells. Suitable prokaryotic cells include eubacteria e.g.enterobacteriaceae such as Escherichia e.g. E. Coli (for example ATCC31, 446; 31, 537; 27, 325), Enterobacter, Erwinia, Klebsiella Proteus,Salmonella e.g. Salmonella typhimurium, Serratia e.g. Serratiamarcescans and Shigella as well as Bacilli such as B. subtilis and B.licheniformis (see DD 266 710), Pseudomonas such as P. aeruginosa andStreptomyces. Of the yeast host cells, Saccharomyces cerevisiae,schizosaccharomyces pombe, Kluyveromyces (e.g. ATCC 16,045; 12,424;24178; 56,500), yarrowia (EP402, 226), Pichia Pastoris (EPI 83, 070, seealso Peng et al J. Biotechnol. 108 (2004) 185-192), Candida, Thehodermareesia (EP244, 234J, Penicillin, Tolypocladium and Aspergillus hostssuch as A. nidulans and A. niger are also contemplated.

Although Prokaryotic and yeast host cells are specifically contemplatedby the invention, preferably however, host cells of the presentinvention are higher eukaryotic cells. Suitable higher eukaryotic hostcells include mammalian cells such as COS-1 (ATCC No. CRL 1650) COS-7(ATCC CRL 1651), human embryonic kidney line 293, baby hamster kidneycells (BHK) (ATCC CRL.1632), BHK570 (ATCC NO: CRL 10314), 293 (ATCCNO.CRL 1573), Chinese hamster ovary cells CHO (e.g. CHO-K1, ATCC NO: CCL61, DHFR: CHO cell line such as DG44 (see Urlaub et al, (1986) SomaticCell Mol. Genet. 12, 555-556)), particularly those CHO cell linesadapted for suspension culture, mouse Sertoli cells, monkey kidneycells, African green monkey kidney cells (ATCC CRL-1587), HELA cells,canine kidney cells (ATCC CCL 34), human lung cells (ATCC CCL 75), HepG2 and myeloma or lymphoma cells e.g. NSO (see U.S. Pat. No. 5,807,715),Sp2/0, YO. Thus in one embodiment of the invention there is provided astably transformed host cell comprising a vector encoding a heavy chainand/or light chain of the therapeutic antibody or antigen bindingfragment thereof as herein described. Preferably such host cellscomprise a first vector encoding the light chain and a second vectorencoding said heavy chain.

4.6.1 Bacterial Fermentation

Bacterial systems may be used for the expression of non-immunoglobulintherapeutic proteins described above. Bacterial systems are alsoparticularly suited for the expression of antibody fragments. Suchfragments are localised intracellular or within the periplasma.Insoluble periplasmic proteins can be extracted and refolded to formactive proteins according to methods known to those skilled in the art,see Sanchez et al (1999) J. Biotechnol. 72, 13-20 and Cu pit P M et al(1999) Lett Appl Microbiol, 29, 273-277.

4.8.7 Cell Culturing Methods.

Host cells transformed with vectors encoding the therapeutic antibodiesof the invention or antigen binding fragments thereof may be cultured byany method known to those skilled in the art. Host cells may be culturedin spinner flasks, roller bottles or hollow fibre systems but it ispreferred for large scale production that stirred tank reactors are usedparticularly for suspension cultures. Preferably the stirred tankers areadapted for aeration using e.g. spargers, baffles or low shearimpellers. For bubble columns and airlift reactors direct aeration withair or oxygen bubbles maybe used. Where the host cells are cultured in aserum free culture media it is preferred that the media is supplementedwith a cell protective agent such as pluronic F-68 to help prevent celldamage as a result of the aeration process. Depending on the host cellcharacteristics, either microcarriers maybe used as growth substratesfor anchorage dependent cell lines or the cells maybe adapted tosuspension culture (which is typical). The culturing of host cells,particularly invertebrate host cells may utilise a variety ofoperational modes such as fed-batch, repeated batch processing (seeDrapeau et al (1994) cytotechnology 15: 103-109), extended batch processor perfusion culture. Although recombinantly transformed mammalian hostcells may be cultured in serum-containing media such as fetal calf serum(FCS), it is preferred that such host cells are cultured in syntheticserum-free media such as disclosed in Keen et al (1995) Cytotechnology17:153-163, or commercially available media such as ProCHO-CDM orUltraCHO™ (Cambrex N.J., USA), supplemented where necessary with anenergy source such as glucose and synthetic growth factors such asrecombinant insulin. The serum-free culturing of host cells may requirethat those cells are adapted to grow in serum free conditions. Oneadaptation approach is to culture such host cells in serum containingmedia and repeatedly exchange 80% of the culture medium for theserum-free media so that the host cells learn to adapt in serum freeconditions (see e.g. Scharfenberg K et al (1995) in Animal Celltechnology: Developments towards the 21st century (Beuvery E. G. et aleds), pp 619-623, Kluwer Academic publishers).

Antibodies or other therapeutic proteins of the invention secreted intothe media may be recovered and purified using a variety of techniques toprovide a degree of purification suitable for the intended use. Forexample the use of therapeutic antibodies of the invention for thetreatment of human patients typically mandates at least 95% purity, moretypically 98% or 99% or greater purity (compared to the crude culturemedium). In the first instance, cell debris from the culture media istypically removed using centrifugation followed by a clarification stepof the supernatant using e.g. microfiltration, ultrafiltration and/ordepth filtration. A variety of other techniques such as dialysis and gelelectrophoresis and chromatographic techniques such as hydroxyapatite(HA), affinity chromatography (optionally involving an affinity taggingsystem such as polyhistidine) and/or hydrophobic interactionchromatography (HIC, see U.S. Pat. No. 5,429,746) are available. In oneembodiment, the antibodies of the invention, following variousclarification steps, are captured using Protein A or G affinitychromatography followed by further chromatography steps such as ionexchange and/or HA chromatography, anion or cation exchange, sizeexclusion chromatography and ammonium sulphate precipitation. Typically,various virus removal steps are also employed (e.g. nanofiltration usinge.g. a DV-20 filter). Following these various steps, a purified(preferably monoclonal) preparation comprising at least 75 mg/ml orgreater e.g. 100 mg/ml or greater of the antibody of the invention orantigen binding fragment thereof is provided and therefore forms anembodiment of the invention. Suitably such preparations aresubstantially free of aggregated forms of antibodies of the invention.

4.9—Screening Methods

In other embodiments, there are provided methods of identifyingmodulators (such as antagonists) capable of modulating the interactionbetween NRG1 (e.g. NRG1β1) and its cognate receptor or receptors (e.g.ErbB2/ErbB3 heterodimer). In some embodiments, the modulator neutralizesthe biological activity of NRG1.

In accordance therefore with the present invention there is provided amethod of screening a candidate compound for its ability to neutralizethe biological activity of NRG1 (particularly NRG1β1) by e.g. inhibitingthe interaction between NRG1 (e.g. NRG1β1) and a cognate receptor suchas ErbB2/ErbB3 heterodimer which method comprises contacting said NRG1(e.g. NRG1β1) with said candidate compound (e.g. a candidate antibody)and detecting a modulation in the interaction between said NRG1 and oneor both of its cognate receptors.

In accordance therefore with the present invention there is provided amethod for screening a candidate compound for its ability to modulate(e.g. inhibit) the interaction between NRG1 (e.g. NRG1β1) and a cognatereceptor (e.g. ErbB2/ErbB3 heterodimer) which method comprisescontacting said NRG1 (e.g. NRG1β1) with said candidate compound (e.g. acandidate antibody) and detecting neutralization of the biologicalactivity of NRG1β1.

In one embodiment, the method comprises detecting a change in MUC5ACand/or MUC5B expression on a cell capable of expressing MUC5AC and/orMUC5B. An example of such a cell is an epithelial cell such as a gobletcell. Methods for detecting changes to such expression will be apparentto the skilled person. Thus in one embodiment of the invention there isprovided a method for screening a candidate inhibitor of the interactionbetween NRG1 (e.g. NRG1β1) and a cognate receptor (such as a ErbB2and/or ErbB3) which method comprises contacting said NRG1 with saidcandidate compound in the presence of a cell expressing MUC5AC and/orMUC5B and detecting a change (e.g. reduction) in expression of MUC5ACand/or MUC5B compared to the expression of said MUC5AC and/or MUC5B onsaid cell in the presence of said NRG1 without said candidate compound.

In another embodiment, the method comprises detecting a change in gobletcell hyperplasia in the presence of said candidate compound. Thus in oneaspect of the invention there is provided a method of/for screening acandidate inhibitor of the interaction between NRG1 (e.g. NRG1β1) and acognate receptor (such as a ErbB2 and/or ErbB3, e.g. ErbB2/ErbB3heterodimer) which method comprises contacting said NRG1 with saidcandidate compound in the presence of a goblet cell and detecting achange (e.g. reduction) in goblet cell division compared to goblet celldivision in the absence of said candidate compound.

4.10 Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising thetherapeutic protein or low molecular weight chemical entity formulatedtogether with a pharmaceutically acceptable carrier. The compositionscan additionally contain other therapeutic agents that are suitable fortreating or preventing a human disease or disorder noted below.Pharmaceutically carriers enhance or stabilize the composition, or tofacilitate preparation of the composition. Pharmaceutically acceptablecarriers include solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible.

A pharmaceutical composition of the present invention can beadministered by a variety of methods known in the art. The route and/ormode of administration vary depending upon the desired results. It ispreferred that administration be intravenous, intramuscular,intraperitoneal, or subcutaneous, or administered proximal to the siteof the target. The pharmaceutically acceptable carrier should besuitable for intravenous, intramuscular, subcutaneous, parenteral,spinal or epidermal administration (e.g., by injection or infusion).Depending on the route of administration, the active compound(particularly low molecular weight chemical entities) may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

The composition should be sterile and fluid. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

Pharmaceutical compositions of the invention can be prepared inaccordance with methods well known and routinely practiced in the art.See, e.g., Remington: The Science and Practice of Pharmacy, MackPublishing Co., 20^(th) ed., 2000; and Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978. Pharmaceutical compositions are preferably manufacturedunder GMP conditions. Typically, a therapeutically effective dose orefficacious dose of the modulator of NRG1 (e.g. NRG1β1) such as a NRG1β1antibody described herein is employed in the pharmaceutical compositionsof the invention. They are typically formulated into pharmaceuticallyacceptable dosage forms by conventional methods known to those of skillin the art. Dosage regimens are adjusted to provide the optimum desiredresponse (e.g., a therapeutic response). For example, a single bolus maybe administered, several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level depends upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors.

A physician can start doses of the antibodies of the invention employedin the pharmaceutical composition at levels lower than that required toachieve the desired therapeutic effect and gradually increase the dosageuntil the desired effect is achieved. In general, effective doses of thecompositions of the present invention, for the treatment of an allergicinflammatory disorder described herein vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Treatment dosages need to be titrated tooptimize safety and efficacy. For administration with an antibody, thedosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5mg/kg, of the host body weight. For example dosages can be 1 mg/kg bodyweight or 10 mg/kg body weight or within the range of 1-10 mg/kg. Anexemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months.

Antibody and other protein therapeutics are usually administered onmultiple occasions. Intervals between single dosages can be weekly,monthly or yearly.

Intervals can also be irregular as indicated by measuring blood levelsof therapeutic protein in the patient. In some methods, dosage isadjusted to achieve a plasma antibody concentration of 1-1000 μg/ml andin some methods 25-300 μg/ml. Alternatively, antibody or other proteintherapeutics can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the antibody or otherprotein therapeutic in the patient. In general, humanized antibodiesshow longer half life than that of chimeric antibodies and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

4.11 Clinical Uses

The present invention is based, at least in part on the finding thatmembers of the neuregulin family (particularly the NRG1 family e.g.NRG1B and in particular NRG1β1) promote goblet cell formation. Thereforemodulators (particularly antagonists) of the present invention may beuseful in the treatment of human diseases or disorders in which aberrantgoblet cell formation plays a pathological role. In accordance thereforethe present invention provides a method of treating a disease ordisorder of goblet cell cycle regulation which method comprisesadministering to a human patient in clinical need thereof atherapeutically effective amount of a modulator of NRG1 biologicalactivity. In some embodiments, the modulator is an antagonist (such asan antibody, particularly a human or humanized antibody of an IgGisotype or an antibody fragment thereof) which binds with either NRG1(e.g. NRG1β1) and/or a cognate receptor thereof (such as ErbB2 and/orErbB3, particularly ErbB2/ErbB3 heterodimer) and inhibits theinteraction there between. In other embodiments there is provided amethod of treating a disease or disorder of goblet cell cycle regulation(for example goblet cell hyperplasia) comprising administering to ahuman patient in clinical need thereof a therapeutically effectiveamount of a therapeutic protein that binds with NRG1β1 (such as a humanor humanized antibody of an IgG1 or IgG4 isotype as herein describedwhich preferentially or specifically binds with NRG1β1). Examples ofclinical diseases or disorders in which goblet cell hyperplasiacontributes include respiratory diseases such as chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), chronic bronchitis,asthma (particularly moderate and severe forms thereof).

Therefore in some embodiments there is provided a method of treating ahuman patient afflicted with a respiratory disease such as COPD, CF,chronic bronchitis or asthma (particularly moderate to severe formsthereof) which method comprises administering to said patient atherapeutically effective amount of a modulator of NRG1 (e.g. NRG1β1)biological activity. In preferred forms of these embodiments, themodulator is an antibody (particularly a human or humanized antibody ofan IgG1 or IgG4 isotype or an antibody fragment that binds with (e.g.preferentially or specifically binds with) NRG1 (particularly NRG1β1)and neutralizes the biological activity of NRG1, particularly NRG1β1. Inthis regard, “biological activity” as used throughout this specificationin reference to NRG1 and in particular NRG1β1 refers to the activity ofthese proteins in promoting the expression of MUC5AC and/or MUC5B. Thus“neutralizes the biological activity” and the like as used throughoutthis specification refers to the inhibition of MUC5B and/or MUC5ACexpression by a modulator of the invention.

In some embodiments, there is provided a method of treating a humanpatient afflicted with a disease or disorder comprising aberrant mucusproduction such as COPD, CF, chronic bronchitis or asthma (particularlythe severe or moderate forms thereof) or a disease or disorder of thelower respiratory tract (e.g. an infection of the lower respiratorytract) which method comprises administering to said patient atherapeutically effective amount of an inhibitor (for example a human orhumanized antibody of a IgG1 or IgG4 isotype) of the interaction betweenNRG1 (particularly NRG1β1) and one or more of its cognate receptors(e.g. ErbB2 and/or ErbB3, particularly ErbB2/ErbB3 heterodimer). Inpreferred forms of these embodiments, the modulator is a human orhumanized antibody or antibody fragment as described herein whichpreferentially or more preferably specifically binds with NRG1β1 andinhibits said aberrant mucus secretion.

In some embodiments, there is provided a method of treating a humanpatient afflicted with a respiratory disease or disorder as COPD, CF,bronchitis (particularly chronic bronchitis) or asthma (particularly thesevere or moderate forms thereof) or a disease or disorder of the lowerrespiratory tract (e.g. an infection of the lower respiratory tract),pneumonia, emphysema which method comprises administering to saidpatient a therapeutically effective amount of an inhibitor (for examplea human or humanized antibody of a IgG1 or IgG4 isotype) of theinteraction between NRG1 (particularly NRG1β1) and one or more of itscognate receptors (e.g. ERB2 and/or ERB3, particularly “ErbB”2/ErbB3heterodimer).

In some embodiments, there is provided a method of treating the aberrantmucus production aspect of a respiratory disease such as COPD, CF,chronic bronchitis or asthma (particularly severe or moderate formsthereof) which method comprises administering to said human patient atherapeutically effective amount of a modulator (such as a human orhumanized antibody) which neutralizes the biological activity of NRG1,and in particular NRG1β.

In other embodiments there is provided a method of treatingprophylactically a human patient at risk of being afflicted with arespiratory disease or disorder characterized by aberrant mucusproduction (such as COPD, chronic bronchitis, cystic fibrosis, asthma)which method comprises administering to said patient a therapeuticallyeffective amount of a modulator (such as therapeutic protein e.g. ahuman or humanized antibody of a IgG isotype e.g. IgG1 or IgG4 as hereindescribed) which modulates the interaction between a NRG1 (particularlyNRG1β and more particularly NRG1β1) and one or more of its cognatereceptor (e.g. ErbB2 and/or ErbB3). In some embodiments, the modulatorneutralizes the biological activity of NRG1.

In other embodiments of the invention there is provided a method oftreating a human patient afflicted with a respiratory disease ordisorder such as COPD, CF, chronic bronchitis which method comprisesco-administering an inhibitor (such as a therapeutic protein e.g. ahuman or humanized antibody as described herein) of NRG I (particularlyNRG113 and more particularly NRG1β1) biological activity together withan anti-human IL-13 agent (such as an IL-13 antibody, particularly ahuman or humanized IL-13 antibody) and/or with an anti-human IL-4 agent(such as an IL-4 antibody, particularly a human or humanized IL-4antibody) and/or with an anti-human IL-5 agent (such as an IL-5antibody, particularly a human or humanized IL-5 antibody) and/or ananti-IgE agent (such as a human or humanized anti-IgE antibody and/oranti-IL-17 (particularly IL-17A) antibody (such as a human or humanizedanti-IL-17, e.g. IL-17A antibody). Each of these combinations isspecifically and separately contemplated.

Although the present invention has been described principally inrelation to the treatment of human diseases or disorders, the skilledperson will readily appreciate that the teaching herein may be appliedto the treatment of similar diseases or disorders in non human mammals.

5. EXEMPLIFICATION

The present invention is now described by way of example only.

5.1 BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Effect of NRG1 beta1 and NRG1 alpha on MUC5AC expression

Human Bronchial Epithelial Cells (HBECs) were treated with increasingconcentrations of NRG1β1 (top left) or NRG1 a (top right) for 7 days andstained with an anti-MUC5AC (45M1) monoclonal antibody. The proportionof MUC5AC positive cells was assessed by image analysis. (bottom)Representative histology images the MUC5AC/alcian blue stained vehicleand NRG1β1 (50 nM) treated HBECs. The arrow indicates an alcian bluestained cell which does not stain with the anti-MUC5AC antibody. Theresults shown are representative results from experiments on 3independent donors. The results shown are the mean±SEM (n=3).Significant differences from the untreated control are indicated(*P<0.05).

FIG. 2: Effect NRG1 alpha and beta1 on MUC5B protein

The effects of increasing concentrations of NRG1β1 (A) and NRG1a (B) onMUC5B protein, as assessed by immunohistochemistry. Cells were treatedwith NRG1 for 7 days. (C) Representative histology images theMUC5B/alcian blue stained vehicle and NRG1β1 (50 nM) treated HBECs. Theresults shown are the mean±SEM (n=3). Significant differences from theuntreated control are indicated (*P<0.05).

FIG. 3: Effects of NRG1 beta1 on MUC5AC and MUC5B expression over atimecourse

The effects of NRG1β1 (50 nM) on MUC5AC protein (A) and MUC5B protein(B) over 1 to 14 days treatment as assessed by histology. NRG1β1 treatedsamples are shown in black and untreated samples in white. The resultsshown are the mean±SEM (n=3). Significant differences from the untreatedcontrol are indicated (*P<0.05).

FIG. 4: NRG1 beta1 expression in primary cells

NRG1β1 expression was analysed by RT-PCR in a panel of primary cellswhich included T-cells, Neutrophils, Human Bronchial Epithelial Cells(HBECs) Bronchial Smooth Muscle Cells (BSMCs) and lung fibroblasts. Theexpression of the housekeeping gene transferrin was also analysed. Datais representative of data obtained with cells from at least 2independent donors.

FIG. 5: MUC5AC and MUC5B expression in lungs of ovalbumin challengedmice

Expression of MUC5AC (A) and MUC5B (B) was analysed by quantitativeRT-PCR in lung tissue from OVA challenged Balb/c mice. Data is shown asmean±SEM (n=8 per group) and is shown normalised to the housekeepinggene β-actin. *p<0.05 compared to saline challenged mice.

FIG. 6: Western blot analysis of NRG1 beta1 protein in BAL fluid fromOVA challenged mice

BAL fluid from OVA challenged mice was analysed for NRG1β1 protein bywestern blot. Blots were probed with an antibody (sc-347) whichrecognizes preferentially the NRG1β1 isoform. BAL fluid from 4 animalswas analysed for each treatment. Data shown are representative of dataobtained with 2 independent sets of samples.

FIG. 7: NRG1 beta1 ELISA

NRG1β1 protein was quantified in BAL fluid from OVA challenged mice.Data is shown as mean±SEM (n=4 mice per group). The data shown isrepresentative of 2 independent sets of samples. **p<0.001 compared tosaline challenged mice.

FIG. 8: Effects of a pan-ErbB receptor inhibitor on goblet cellformation

The effects of a pan-ErbB receptor inhibitor on NRG1β1-induced gobletcell formation are shown. The effects of inhibitor (1-10 μM) onNRG1β1-induced MUC5AC and MUC5B protein were determined by histology.Cells were either untreated or treated with NRG1β1 (50 nM) as indicated.*Significant difference from the untreated control (P<0.05); #significant difference from the NRG1β1 only treated group (P<0.05); ##significant difference from the NRG1β1 only treated group (P<0.001). Theresults are shown as the mean±SEM (n=3) and are representative of 2independent experiments.

FIG. 9: Analysis of ErB receptor expression in Human BronchialEpithelial Cells

ErbB receptor expression in primary human bronchial epithelial cells wasanalysed by RT-PCR (left) and western blot (right). ErbB receptorexpression was analysed in 3 or 2 donors for mRNA and protein,respectively. Expression of the housekeeping gene GAPDH is also shown.

FIG. 10: Effects of ErbB receptor neutralising antibodies onNRG1β1-induced goblet cell formation

Effects of anti-ErbB receptor antibodies on NRG1β1-induced MUC5ACprotein (A, C, E) and MUC5B protein (B, D, F) in HBECs were quantifiedby histology. Data for an anti-ErbB2 receptor antibody (A, B),anti-ErbB3 receptor antibody (C, D) and an anti-ErbB4 receptor antibody(E, F) are shown and the concentration of antibody used are indicated onthe graphs. *Significant difference from the untreated control (P<0.05);# significant difference from the NRG1β1 only treated group (P<0.05).The results are shown as the mean±SEM (n=3) and are representative of 2independent experiments.

5.2 LIST OF ABBREVIATIONS

Abbreviation Description ALI Air Liquid Interface BAL Bronchial AlveolarLavage BSA Bovine Serum Albumin BSMC Bronchial Smooth Muscle Cell CFCystic Fibrosis COPD Chronic Obstructive Pulmonary Disease ELISAEnzyme-Linked Immunosorbent Assay HBEC Human Bronchial Epithelial CellsHRG Heregulin IL-13 Interleukin 13 MUC5AC Mucin 5AC MUC5B Mucin 5B NRG1Neuregulin-1 OVA Ovalbumin PAS Periodic Acid-Schiff RT-PCR ReverseTranscriptase Polymerase Chain Reaction TMB3,3′,5,5′-Tetramethylbenzidine EGFR Epidermal Growth Factor ReceptorErbB2 v-erb-b2 avian erythroblastic leukaemia viral oncogene homolog 2

5.3 Methods 5.3.1 Culture of HBECs

Human Bronchial epithelial cells (HBECs; Cambrex) were cultured inbronchial epithelial cell growth medium (BEGM; Cambrex) supplementedwith the provided singlequots essentially as described in [Atherton etal, 2003]. For differentiation, cells were grown on 0.4 μM pore size, 12mm Transwell inserts (Costar) at a cell density of 8.25×10⁴ cells/insertin differentiation medium. Differentiation medium contained 50% BEGM and50% Dulbeccos' modified Eagle's medium (DMEM) and was supplemented with52 μg/ml bovine pituitary extract, 5 μg/ml insulin, 0.5 μg/mlhydrocortisone, 10 μg/ml transferrin, 0.5 μg/ml epinephrine 0.5 μg/mlhuman EGF, 50 μg/ml gentamicin and 50 nM retinoic acid. Cells weremaintained submerged for 7 days then grown at air-liquid interface (ALI)for 7-14 days and medium was replenished every 2-3 days. Cells weretreated with NRG1a or NRG1β1 (R&D systems) during the ALI cultureperiod, added to the basolateral chamber of the Transwell inserts. Atall stages cells were maintained at 37° C. in the presence of 5% CO₂ inan air incubator.

5.3.2. Immunohistochemical Detection of MUC5AC and MUC5B Protein

After 7 days (unless otherwise stated) of differentiation at ALI, theapical surface of the HBECs was washed gently with PBS and fixed withneutral buffered formalin and wax embedded. Inserts were sectioned at 3μm thickness and stained with either an anti-MUC5AC antibody (45M1;Labvision) or anti-MUC5B monoclonal antibodies using a DABMAP protocolon a Ventana XT immunostainer and counterstained with 1% alcian blue in3% acetic acid, pH 2.5. The area of staining was assessed using a ZeissAxioplan 2 microscope (×10 magnification) with an Imaging AssociatedKS400 image analyzer (Imaging Associates). Fourteen fields were scoredfor each sample. Data are presented as goblet cell density, which wasdefined as the ratio of stained area (μm²) to length (μm) of epitheliumscored. Custom made anti-MUC5B monoclonal antibodies were obtained fromthe Hybridoma Core Laboratory, University of Florida and were raisedagainst the peptide SWYNGHRPEPGLG (SEQ.I.D.NO:11).

5.3.3 Preparation of RNA and First Strand cDNA Synthesis

Total RNA was isolated from primary cells as described previously [Joneset al, 2003]. Total RNA was isolated from cells using the RNeasy miniRNA isolation kit (Qiagen) according to the manufacturer's instructions.RNA was also isolated from lung tissue from the mouse ovalbumin (OVA)model of allergen-induced goblet cell formation. Balb/c mice had beensensitized with OVA over 2 weeks and given a daily challenge ofovalbumin (50 mg/ml) for 2 consecutive days, as described in [Trifilieffet al, 2000]. RNA was prepared from mouse lung tissue using the reagentsand protocols supplied in the Qiagen RNeasy miniprep kit. Frozen lungtissue (20 mg) was homogenized in 600 μl of RLT buffer using a polytronhomogenizer (Kinematica AG). Lysates were further processed usingQiashredder columns (Qiagen) according to the manufacturer'sinstructions.

First strand cDNA synthesis was performed using 1 μg of total RNA andthe reagents and protocol provided in the first strand cDNA synthesiskit (Roche Diagnostics Ltd.).

5.3.4 Quantitative RT-PCR

MUC5AC and MUC5B gene expression in lung tissue from the mouse OVA model(Section 2-3) was analysed by quantitative RT-PCR. PCR reaction mixtureswere prepared in a final volume of 20 μl and contained 10 μl 2×SYBRGreen PCR Master Mix (Sigma), 0.8 μl of 10 μM of each forward andreverse primer (final concentration 400 nM) and 4.4 μl dH₂O, Sixteen μlof this master mix was placed into each well of a 96-well OpticalReaction Plate and 4 μl of cDNA template (Section 2-3) was added to eachwell. Samples were analysed in duplicate using an ABI7900 SequenceDetection System (Applied Biosystems). Primer sequences for MUC5AC,MUC5B and the β-actin housekeeping gene control are shown in Table 1.

A standard curve was included on each plate using a mixed lung cDNA poolwith cDNA concentrations in the range 66,666 to 274 pg. A no templatecontrol (NTC) was included which contained nuclease free water insteadof diluted cDNA The quantitative PCR program was as follows: Stage 1,50° C. for 2 min; Stage 2, 95° C. for 10 min; Stage 3, 40 cycles of 95°C. for 15 s, 60° C. for 15 s and 72° C. for 30 s. Data were interpretedby the relative method (ABI PRISM 7700 Sequence Detection System. UserBulletin #2, PE Applied Biosystems, 1997). Expression values are shownnormalised to the housekeeping gene β-actin.

TABLE 1 RT-PCR primers Length of Gene Primer sequences (5′ to 3′)Primer names PCR product (bp) Transferrin TTACAGTGGCTGTATTCTGCTGGTransFor 401 (SEQ. I.D. NO: 1) TGCTGTTCTCATGGAAGCTATGG TransRev(SEQ. I.D. NO: 2) NRG1β1 CAAGCATCTTGGGATTGAA NRG1β1For 188(SEQ. I.D. NO: 3) TGTTTCGTTCTGACCGAAGG NRG1Rev (SEQ. I.D. NO: 4) Muc5acCAGCCGAGAGGAGGGTTTGATCT Muc5acFor 399 (SEQ. I.D. NO: 5)AGTCTCTCTCCGCTCCTCTCAAT Muc5acRev (SEQ. I.D. NO: 6) Muc5bAGGAAGACCAGTGTGTTTGTC Muc5bFor 615 (SEQ. I.D. NO: 7)GTCCTCATTGAAGAAGGGCTG Muc5bRev (SEQ. I.D. NO: 8) β-actinTGTGATGGTGGGAATGGGTCAG MmβactinFor 514 (SEQ. I.D. NO: 9)TTTGATGTCACGCACGATTTCC MmβactinRev (SEQ. I.D. NO: 10)

5.3.5 RT-PCR

For analysis of NRG1β1 gene expression in cDNA prepared from primaryhuman cells. PCR reactions contained 10 μl of 2× HotStar Taq Master Mix(Qiagen), 20 pmol each of NRG1β1For and NRG1Rev primers (Sigma-Genosys;Table 1), 1 μl of cDNA template (50 ng) made up in a final volume of 20μl in 0.2 ml thin walled PCR tubes. Control PCR reactions were performedwith primers specific to the housekeeping gene transferrin using primersTransFor and TransRev (Table 1). PCR cycling conditions were as follows:Denaturation at 95° C. for 15 min, 30 cycles of denaturation 94° C. for15 s, annealing at 55° C. for 15 s, and extension at 72° C. for 45 s,followed by a final extension of 5 min at 72° C. PCR products wereanalysed on 2% agarose gels and stained with ethidium bromide.

5.3.6 Western Blotting

Western blot analysis was performed on bronchoalveolar lavage (BAL)samples from the mouse OVA model. Samples containing 25 μA of BAL fluidwere denatured for 10 min at 70° C. in 1× NuPAGE LDS sample buffer, 50mM DTT prior to analysis on 4-12% NuPAGE Bis-Tris acrylamide gels at 200V using the NuPAGE MOPS running buffer. Gels and buffers were purchasedfrom Invitrogen. Samples were transferred onto Immobilon-P PVDF membrane(Millipore) using NuPAGE transfer buffer (Invitrogen) at 10 V overnight.Membranes were blocked in PBS, 0.1% (v/v) tween-20, 5% (w/v) Blotto milkpowder (Santa Cruz) for 1 h at room temperature. Blocking buffer wasremoved and membranes incubated at room temperature for 3 h withanti-NRG1β1 (sc-347; Santz Cruz) primary antibody diluted 1:600 inblocking buffer. Membranes were washed 4 times for 10 min in wash buffer(PBS, 0.1% (v/v) tween-20) at room temperature with shaking. Blots wereincubated in the dark for 1 h with shaking with goat anti-rabbitIRDye800 secondary antibody (Tebu-bio) diluted 1:2000 in 0.5×PBS, 0.05%(v/v) tween-20, 0.5× Odyssey blocking buffer, 0.01% (w/v) SDS. Membraneswere washed as described previously and analysed using a LI-COR OdysseyInfrared imaging system (LI-COR Biosciences) at 169 μm resolution, focusoffset of 3.0 mm and intensity setting of 5.0. NRG1a or NRG1β1 controlproteins (Labvision) were included as positive controls.

5.3.7 NRG1 beta1 ELISA

NRG1β1 protein levels in BAL fluid of OVA challenged mice (Section 2-3)were analysed using the NRG1β1 DuoSet ELISA kit (R&D systems) using thereagents supplied in the kit. Immuno plates (96-well; Nunc) were coatedovernight at room temperature with 100 μl/well mouse anti-human NRG1β1at 4 μg/ml. Plates were washed 4 times with 400 μl/well of wash buffer(PBS, 0.05% (v/v) tween-20). Plates were blocked for 1 h at roomtemperature with 300 μl/well of buffer A (PBS, 1% (w/v) BSA), thenwashed as described previously. BAL fluid samples (100 μl/well) orNRG1β1 protein standard diluted in buffer A (0.0625-4 ng/ml) were addedto the wells and incubated for 2 h at room temperature. A blankcontaining buffer A was also included on the plate. The plate was washedas described previously before adding 100 μl/well of detection antibody,biotinylated goat anti-human NRG1β1, at 200 ng/ml for 2 h at roomtemperature. The plate was washed again and 100 μl/well streptavidinconjugated to horseradish peroxidase (100 μl/well) was added at 1:200dilution in buffer A for 30 min at room temperature.3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sigma; 100 μl/well) wasadded to the plate and incubated for 30 min at room temperature. Thereaction was stopped by adding 50 μl/well of 1 M sulphuric acid. Theabsorbance at 450 nm, corrected for background measured at 540 nm, wasmeasured using a Spectramax 340 plate reader (Molecular Devices).Samples were analysed in duplicate.

5.3.8 Data Analysis and Statistics

Data is shown as the mean±SEM and represents duplicate samples from 2independent experiments. Two sample t-tests were performed to determineif there were significant differences between control and treatmentgroups (*P<0.05, **P<0.001).

5.4 Results 5.4.1 Effects of NRG1 Alpha and Beta1 on Mucin Expressionand Goblet Cell Formation

In cultures of primary human bronchial epithelial Cells (HBECs) grown atALI NRG1β1 treatment caused a dose-dependent increase in MUC5AC protein,a marker for airway goblet cells, as assessed by histology (FIG. 1A). At50 nM NRG1β1, a 3-fold increase in MUC5AC protein over vehicle levelswas obtained. A closely related NRG1 isoform, NRG1a, did not have asignificant effect on MUC5AC protein at concentrations of up to 250 nM(FIG. 1B). HBEC inserts stained with the MUC5AC antibody were alsocounterstained with alcian blue. Interestingly, several alcian bluestained cells which did not stain with the MUC5AC (45M1) antibody werenoted in the NRG1β1 treated samples (FIG. 1C), which might reflect thepresence of other mucins such as MUC5B. To confirm any effects of NRG1β1and NRG1a on MUC5B protein, cells were also stained with a monoclonalantibody against MUC5B. Both NRG1 isoforms caused a dose-dependentincrease in MUC5B protein up to a concentration of 50 nM (FIG. 2). At 50nM, NRG1β1 and NRG1a caused a 9.8 and 6.4-fold increase in MUC5B proteinrespectively. As the NRG1β1 isoform had the most pronounced effects onMUC5AC and MUC5B protein, further studies focused on the NRG1β1.

The effects of NRG1β1 on MUC5AC and MUC5B protein expression over atimecourse is shown in FIG. 3. Expression of MUC5AC and MUC5B proteinwas significant compared to vehicle treated controls after 7, 10 or 14days of NRG1β1 treatment. (FIG. 3).

The gene expression profile of NRG1 μl was analysed in several primarycells. NRG1β1 was found to be expressed in differentiated primary humanbronchial epithelial cells (HBECs), bronchial smooth muscle cells (BSMC)and lung fibroblasts (FIG. 4). No expression was detected in T-cells orneutrophils.

Lung tissue from mice challenged with OVA or saline was analysed byquantitative RT-PCR for MUC5AC and MUC5B gene expression. A 57-foldincrease in MUC5AC gene expression was observed after OVA challenge(FIG. 5) compared to saline controls. MUC5B gene expression alsoincreased 3-fold after OVA challenge (FIG. 5).

NRG1β31 protein expression in BAL fluid from OVA challenged mice wasanalysed by western blot. An increase in NRG1β1 protein was detected inBAL fluid from OVA challenged mice compared to saline challenged controlmice (FIG. 6). NRG1 μl protein in BAL fluid of OVA challenged mice wasquantified by ELISA. A 9-fold increase in NRG1β1 was detected in BALfluid of OVA challenged mice compared to saline challenged control mice(FIG. 7), consistent with the western blot data. Concentrations of NRG1μl were around 0.9 ng/ml.

5.4.2 Discussion

The mucins MUC5AC and MUC5B are the predominant component of airwaysecretions in patients with asthma and COPD [Rose and Voynow, 2006;Rogers, 2003]. In the airways of asthmatics, COPD and CF patientsincreased numbers of goblet cells have been reported [Aikawa et al,1992; Ordonez et al, 2001; Saetta et al, 2000; Gronenberg et al, 2002b].Increased numbers of goblet cells in the airways can be modeled in themouse OVA model of antigen-induced inflammation. A single or repeatedchallenged of OVA results in an increase in alcian blue/PAS stainedgoblet cells in the airways [Trifilieff, El-Hasim and Bertrand, 2000].We have shown in this report a significant increase in expression ofmucins genes MUC5AC and MUC5B in mouse lungs after OVA challenge. Thisdata is consistent with published data where an increase in MUC5AC geneexpression in the OVA model was detected by northern blot analysis[Zhudi Alimam et al, 2000].

We have shown that NRG1β1 induces MUC5AC and MUC5B protein expression indifferentiated HBEC cultures, both markers of airway goblet cells.NRG1β1 rather than the closely related isoform NRG1 a, had the mostpronounced effect on MUC5AC and MUC5B in the in vitro model of gobletcell formation. To further understand the role of NRG1β1 in goblet cellformation in vivo, NRG1β1 protein was analysed in BAL fluid from OVAchallenged mice. An increase in NRG1β1 protein was detected by westernblot and quantified by ELISA, accompanied by increases in MUC5AC andMUC5B gene expression in the lungs of OVA challenged mice. NRG1 haspreviously been shown to stimulate differentiation of human airwayepithelia and to cause an increase in goblet cell number in human airwayepithelial cultures [Vermeer et al, 2006]. However, the NRG1β1 isoformwas not investigated in the study of Vermeer et al. and no effects onthe mucins MUC5AC and MUC5B reported. NRG1β1 was found in this currentstudy to be expressed in lung-derived cells which included bronchialepithelial cells, bronchial smooth muscle cells and lung fibroblasts.

Thus we have shown that the mucin genes MUC5AC and MUC5B are increasedin lungs of OVA challenged mice and this is accompanied by an increasein NRG1β1 protein in the airways. We have also shown that NRG101 ispotent mediator of MUC5AC and MUC5B-positive goblet cells in vitro andtherefore NRG1β1 may represent a potential therapeutic target forrespiratory diseases such as asthma, COPD and CF where mucushypersecretion plays a role.

5.5—Interaction Between NRG1β1 and ErbB 5.5.1 Methods 5.5.1.1 Compoundand Antibody Treatment of Cells

Compounds or antibodies were diluted in HBEC differentiation medium andadded to the basolateral chamber of HBECs grown on transwell inserts 2 hprior to the addition of NRG1β1. HBECs were treated with compound orantibody in combination with NRG1β1 for 7 days at ALI, replacing theNRG1β1 together with fresh antibody or compound every time the mediumwas replenished (every 2-3 days). For each treatment at least 3identical wells were prepared. ErbB2 receptor (AF1129) and ErbB3receptor antibodies (MAB3841) were from R&D systems and ErbB4 receptorantibody (MS-304-PIABX) was from Lab Vision Corporation.

5.5.1.2 RT-PCR Analysis of ErbB Receptor Expression

For analysis of ErbB receptor expression in cDNA prepared fromdifferentiated HBECs, PCR reactions were prepared as follows: PCRreaction mixtures contained 12.5 μl HotStar Taq Master mix (Qiagen), 50pmol of each forward and reverse primer (Table 2), 50 ng cDNA and waterto a final volume of 25 μl in 0.2 ml thin-walled PCR tubes. Control PCRreactions were performed with primers specific to the housekeeping geneGAPDH using primers GAPDHF and GAPDHR (Table 2). PCR cycling conditionswere as follows: Denaturation at 95° C. for 15 min, 35 cycles ofdenaturation 94° C. for 15 s, annealing at 55° C. for 15 s, andextension at 72° C. for 45 s, followed by a final extension of 5 min at72° C. PCR products were analysed on 2% agarose gels and stained withethidium bromide.

TABLE 2 RT-PCR primers for analysis of ErbB receptor expressionLength of Primer sequences Primer PCR product Gene (5′ to 3′) names (bp)ErbB1 gtcctcattgccctcaacacag ErbB1F 326 (SEQ. I.D. NO: 12)ccattgggacagcttggatcac ErbB1R (SEQ. I.D. NO: 13) ErbB2cagttaccagtgccaatatcc ErbB2F 250 (SEQ. I.D. NO: 14) ttgtgcagaattcgtccccErbB2R (SEQ. I.D. NO: 15) ErbB3 actctgaatggcctgagtg ErbB3F 253(SEQ. I.D. NO: 16) caaacttcccatcgtagacc ErbB3R (SEQ. I.D. NO: 17) ErbB4ACCAGCATTGAGCACAACC ErbB4F 368 (SEQ. I.D. NO: 18) CGTCCACATCCTGAACTACCErbB4R (SEQ. I.D. NO: 19) GAPDH CCACCCATGGCAAATTCCATGGCA GAPDHF 598(SEQ. I.D. NO: 20) TCTAGACGGCAGGTCAGGTCCACC GAPDHR (SEQ. I.D. NO: 21)

5.5.1.3 Western Blot Analyses of ErbB Receptor Expression

Cells were lysed in ice-cold lysis buffer which contained 50 mM Tris pH7.5, 150 mM NaCl, 0.65% v/v NP-40 supplemented with Complete proteaseinhibitor cocktail (Roche) and lysates cleared by centrifugation at16000×g for 5 min at 4° C. Protein concentration was determined using aMicro BCA Protein Assay Kit (Perbio) according to the manufacturer'sinstructions. Cleared lysates were denatured at 70° C. for 10 min in 1×NuPAGE sample buffer (Invitrogen) and equal amounts of protein fromsamples resolved using Bis-Tris NuPAGE polyacrylamide gels with MOPSrunning buffer (Invitrogen). Proteins were transferred to Immobilon-PPVDF membranes (Millipore) in NuPAGE transfer buffer (Invitrogen).Primary antibodies were used at 1/1000 dilution and appropriatesecondary antibodies at 1/2000 or 1/5000 dilution. All primaryantibodies were from Santa Cruz Biotechnology and were rabbit polyclonalantibodies, except where indicated as follows: EGFR (1005); ErbB2(C-18), ErbB3 (C-17), ErbB4 (C-18) and GAPDH, a mouse monoclonal primaryantibody (6C5). The secondary antibodies were Alexa Fluor 680 conjugatedanti-rabbit secondary antibody (Invitrogen) or IRDye 800 conjugatedanti-mouse secondary antibody (Tebu-bio). Membranes were incubated inblocking buffer (PBS containing 0.1% v/v tween-20 and 5% w/v Blotto(Santa Cruz) for 4 h at room temperature followed by overnightincubation at 4° C. with primary antibodies diluted in blocking buffer.Membranes were washed with washing buffer (PBS, 0.1% v/v tween-20)before incubation with respective infrared-dye conjugated secondaryantibodies diluted in Odyssey blocking buffer (50% v/v PBS: 50% v/vOdyssey buffer (LI-COR Biosciences UK Ltd) supplemented with 0.1% v/vtween-20 and 0.01% w/v SDS at room temperature for 1 h in the dark.Following incubation with the secondary antibodies, membranes were againwashed in the dark before being imaged using a LI-COR Odyssey infraredimaging system at 169 μm resolution, focus offset of 3.0 mm andintensity setting of 5.0.

5.6 Results

5.6.1 Effects of a pan-ErbB Receptor Inhibitor on Goblet Cell Formation

The effects of a pan-ErbB receptor inhibitor on NRG1β1-induced gobletcell formation were analyzed. HBECs were pre-treated with inhibitorprior to addition of NRG1β1 and cells were then grown for 7 days at ALI.The effects on the goblet cell markers MUC5AC and MUC5B were analyzed byhistology. The NRG1β1-induced increases in MUC5AC and MUC5B protein weresignificantly inhibited by the pan-ErbB receptor inhibitor at 10 μM(FIG. 8).

5.6.2 Effects of Anti-ErbB Receptor Antibodies on NRG1β1-Induced GobletCell Formation

The ErbB receptor expression profile was analysed in HBECs from multiplehuman donors. Protein and mRNA for all four ErbB receptor family membersEGFR, ErbB2, ErbB3 and ErbB4 could be detected in samples from multipleprimary human bronchial epithelial cell donors (FIG. 9).

The ErbB receptor involved in NRG1β1-induced goblet cell formation wasassessed using neutralising antibodies against either ErbB2, ErbB3 orErbB4 receptors in the primary human bronchial epithelial cell model ofgoblet cell formation. HBECs were pre-treated with antibody prior toNRG1β1 treatment at ALI for 7 days. Antibodies against the ErbB2 andErbB3 receptors inhibited NRG1β1-induced MUC5AC and MUC5B production,whereas an antibody against ErbB4 had no effect (FIG. 10).

5.6.3 Discussion

NRG1β1 has been reported to signal through ErbB2/ErbB3 or ErbB2/ErbB4heterodimers [Falls, 2003; Citri et al, 2003]. Expression analysesindicated that all of the ErbB receptor family members, EGFR,ErbB2-ErbB4 were expressed on primary human bronchial epithelial cells.To further delineate which receptors are involved in NRG1β1-inducedgoblet cell formation, a pan-ErbB receptor tyrosine kinase inhibitor[Traxler et al, 2004] was analysed in an in vitro goblet cell formationassay. The pan-ErbB receptor tyrosine inhibitor significantly reducedexpression of the goblet cell mucins MUC5AC and MUC5B, indicating ErbBreceptor involvement in this process. To further dissect which ErbBreceptor was responsible for the NRG1β1-induced goblet cell formation,neutralising antibodies against the ErbB2, ErbB3 and ErbB4 receptorswere tested. Neutralising antibodies against ErbB2 and ErbB3significantly reduced NRG1β1-induced goblet cell formation, as indicatedby the goblet cell markers MUC5AC and MUC5B. In contrast, the anti-ErbB4receptor neutralising antibody had no effect on NRG1β1-induced gobletcell formation. This data suggests that the ErbB2 and ErbB3 receptorsare involved in NRG1β1-induced goblet cell formation in human airwayepithelial cells.

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1. A modulator of NRG1 which modulates or inhibits the interactionbetween NRG1 and a cognate receptor to inhibit goblet cell hyperplasia.2. The modulator of claim 1 wherein the NRG1 is NRG1α or a member of theNRG1β family.
 3. The modulator of claim 2 wherein the member of theNRG1β family is NRG1β1.
 4. The modulator of claim 1 wherein the NRG1 isthe membrane bound and/or soluble form of NRG1.
 5. The modulator ofclaim 1 wherein the modulator is a low molecular weight chemical entity.6. The modulator of claim 1 wherein the modulator is an antibody.
 7. Themodulator of claim 6 wherein the antibody is a monoclonal.
 8. Themodulator of claim 7 wherein the antibody is a human, humanized orchimeric antibody.
 9. The antibody of claim 6 wherein the antibody bindswith NRG1 and modulates or inhibits the interaction between the NRG1 andone or more of its cognate receptors.
 10. The antibody of claim 9wherein the antibody preferentially binds with NRG1β1 (compared withNRG1α.
 11. The antibody of claim 9 wherein the antibody specificallybinds with NRG1β1 compared to NRG1α.
 12. The antibody of claim 6 whereinthe antibody comprises a human constant region of IgG1 or IgG4.
 13. Useof a modulator of claim 1 in the manufacture of a medicament for thetreatment of a disease or disorder featuring aberrant mucus production.14. Use of claim 13 wherein the disease or disorder is respiratory. 15.Use of claim 14 wherein the disease or disorder is COPD, cystic fibrosis(CF), chronic bronchitis, asthma.
 16. Use of claim 13 wherein thedisease or disorder is human.
 17. Use of the modulator of claim 1 in themanufacture of a medicament for the prevention or slowing the onsetand/or ameliorating the symptoms of a disease featuring aberrant mucusproduction.
 18. A method of treating a human patient afflicted with adisease or disorder featuring aberrant mucus production, wherein themethod comprises the step of: administering to said patient atherapeutically effective amount of a modulator of claim
 1. 19. A methodof identifying a modulator of claim 1 which method comprises; (a)providing a candidate modulator; (b) contacting said modulator with saidNRG1; (c) determining whether said candidate modulator modulates orinhibits the interaction between the NRG1 of with its cognate receptor,wherein the ability of the candidate modulator to modulate or inhibitthe interaction between NRG1 and its cognate receptor is correlated withthe ability of the modulator to inhibit goblet cell hyperplasia.
 20. Apharmaceutical composition comprising a modulator of claim 1 togetherwith a pharmaceutically acceptable carrier.
 21. A method of treating ahuman patient afflicted with a respiratory disease or disordercharacterized by aberrant mucus production which method comprises thestep of: co-administering an inhibitor that inhibits the interactionbetween NRG1 and ErbB2, ErbB3, and/or the ErbB2/ErbB3 heterodimer,together with: one or more agents selected from the group consisting of:an anti-human IL-13 agent which inhibits the interaction between humanIL-13 and its cognate human receptor; an anti-human IL-4 agent whichinhibits the interaction between human IL-45 and its human IL-4receptor; an anti-human IL-5 agent which inhibits the interactionbetween human IL-5 and its cognate receptor; and/or an anti-IgE agentwhich inhibits the interaction between human IgE and its cognatereceptor.
 22. The method of claim 21 wherein the NRG1 is NRG1β1.
 23. Themethod of claim 21 wherein the inhibitor that inhibits the interactionbetween NRG1 and the ErbB2/ErbB3 heterodimer is an antibody that bindsto NRG1.
 24. The method of claim 21 wherein the respiratory disease ordisorder characterized by aberrant mucus production is selected from thegroup consisting of: COPD, cystic fibrosis (CF), and chronic bronchitis.25. The method of claim 21 wherein the inhibitor inhibits theinteraction between NRG1 and the ErbB2/ErbB3 heterodimer.
 26. Apharmaceutical composition comprising a modulator of claim 1 togetherwith a pharmaceutically acceptable carrier for the treatment of COPD,cystic fibrosis (CF), asthma, chronic bronchitis, diseases of the lowerrespiratory tract such as respiratory infections, emphysema, pneumonia.27. The modulator of claim 1, wherein the cognate receptor is selectedfrom the group consisting of: ErbB2, ErbB3, and ErbB2/3 heterodimer. 28.The antibody of claim 9, wherein the one or more cognate receptors isselected from the group consisting of: ErbB2, ErbB3, and ErbB2/3heterodimer.
 29. The use of claim 13, wherein the disease or disorderfeaturing aberrant mucus production is in a human patient.
 30. The useof claim 17, wherein the disease featuring aberrant mucus production isselected from the group consisting of: CORD, cystic fibrosis (CF),chronic bronchitis, and asthma.
 31. The method of claim 18, wherein thedisease featuring aberrant mucus production is selected from the groupconsisting of: COPD, cystic fibrosis (CF), chronic bronchitis, andasthma.
 32. The method of claim 19, wherein the cognate receptor isselected from the group consisting of: ErbB2, ErbB3, and ErbB2/3heterodimer.